Peptide libraries with non-canonical amino acids

ABSTRACT

including stereoisomers, pharmaceutically acceptable salts and prodrugs thereof, wherein R, R1, L1, L2, G, M, Y1 Y2 and SEQ are as defined herein. Methods associated with preparation and use of such peptides, as well as pharmaceutical compositions comprising such peptides, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/755,403, filed Nov. 2, 2018. Application No. 62/755,403, filed Nov.2, 2018, is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of peptide librariesand specifically in the area of peptide libraries for design andselection of protein binding agents.

BACKGROUND OF THE INVENTION

Detection of disease at the earliest stages requires multiplexmeasurements of key protein biomarkers in biological samples. Theavailability of high-affinity, highly selective compositions thatrecognize biomarkers from complex biological mixtures is a criticalcomponent for accurate detection of proteins that may indicate diseaseor changes in health. Peptide affinity agents have been suggested foruse as agents for in vitro and/or in vivo detection of disease causingproteins.

Peptide affinity agents that bind to various targets (e.g. proteins) maybe identified by screening large peptide libraries, and then usingvarious techniques to identify which peptide library elements exhibitthe desired interaction with the target. Those peptide libraries may bebiologically synthesized (e.g. bacterial or viral phage display), orthey may be chemically synthesized (e.g. one-bead-one-compound (OBOC)libraries). For chemically synthesized libraries, a candidate peptidebinder is often first identified using a chemical label. For example, ifa protein binds to a particular peptide sequence on a particular bead,then labeling that protein with a fluorescent molecule, or using asimilarly labeled antibody to detect the bead-bound protein, can be usedto identify the bead that contains the peptide of interest.

Regardless of their preparation method, the sequence of the peptide ofinterest must then be determined. Typical methods for determining thatsequence include mass spectrometric sequencing, or Edman degradation.Thus, peptide libraries for identification of protein affinity agentsare preferably readily sequencable by common techniques.

The scope of amino acids, and thus of the physical and chemical space ofthe peptides, has generally been limited to the natural amino acids. Abroader or different scope of physical and chemical properties ofpeptides could provide additional or better protein targeting peptides.

Accordingly, there is a need for improved protein targeting peptides. Inparticular, there is a need for peptides having a broader or differentscope of physical and chemical properties. The present inventionfulfills this need and provides further related advantages.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is not to betaken as an admission that any or all of these matters form part of theprior art base or were common general knowledge in the field relevant tothe present disclosure as it existed before the priority date of eachclaim of this application.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

BRIEF SUMMARY OF THE INVENTION

Disclosed are peptides and libraries of peptides comprising amino acidsselected from a set of amino acids that includes both canonical andnon-canonical amino acids. In some forms, the amino acid set can includetwo or more amino acids from each of the categories (a) hydrophobic sidechain—aliphatic, (b) hydrophobic side chain—aromatic, (c) polar sidechain—neutral, (d) polar side chain—charged, and (e) conformationalperturbation.

In some forms, the amino acid set can include two or more amino acidsfrom each of the categories (a) hydrophobic side chain—aliphatic, (b)hydrophobic side chain—aromatic, (c) polar side chain—neutral, and (d)polar side chain—charged, and can include one or more amino acids fromthe conformational perturbation category.

In some forms, the amino acid set can comprise 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids. Insome forms, the amino acid set can consist of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

In some forms, the amino acid set can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26different non-canonical amino acids.

In some forms, the amino acid set can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different canonicalamino acids.

In some forms, the amino acid set can comprise any combination ofcanonical amino acids and non-canonical amino acids, wherein at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofthe amino acids in the amino acid set is a non-canonical amino acid andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 of the amino acids in the amino acid set is a canonical aminoacid, and wherein the set of amino acids totals 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

In some forms, the amino acid set can consist of any combination ofcanonical amino acids and non-canonical amino acids, wherein at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofthe amino acids in the amino acid set is a non-canonical amino acid andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 of the amino acids in the amino acid set is a canonical aminoacid, and wherein the set of amino acids totals 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

Preferably, the disclosed peptides are cyclic peptides having activityas protein affinity agents, including stereoisomers, pharmaceuticallyacceptable salts and prodrugs thereof, and methods for identificationand use of the same.

Advantageously, embodiments of the cyclic peptides described herein canbe prepared from natural and/or non-natural amino acids (i.e., canonicaland/or non-canonical amino acids) using common solid-state peptidesynthesis techniques and can be readily sequenced. In variousembodiments, the cyclic peptides comprise a variable region (VR) andconstant region (CR). The variable region generally comprises a peptidesequence which varies among peptides in a library, thus providing ameans for identifying a sequence having affinity for a desired target,such as a protein epitope. The constant region, in various embodiments,comprises functionality to aid in the screening process, such as:chemical groups that aid in sequencing, chemical groups that providehandles for various assays, chemical groups that are important forclosing the peptide into a cycle (e.g., triazole, carbon-carbon doublebonds), chemical groups that provide additional biochemical or chemicalproperties (stability, cell wall penetration, etc.) reporter groups(e.g., bimolecular labels such as biotin) and/or other useful chemicalmoieties. In certain embodiments, the CR comprises an exocyclic aminegroup which provides a means for sequencing the peptides via standardEdman degradation.

In some forms, the peptide is a cyclic peptide having the followingstructure (I):

or a salt, tautomer, prodrug or stereoisomer thereof, wherein R, R¹, L¹,L², G, M, Y¹ Y² and SEQ are as defined herein. In particular, disclosedare such peptides having one or more non-canonical amino acids in SEQ.Compositions comprising the cyclic peptide structure (I) and librariescomprising a plurality of the cyclic peptides are also provided in otherembodiments.

In another embodiment, a method for identifying a target binding agentcompound is also provided, the method comprising:

A) providing a first peptide library comprising a plurality of firstpeptide library members, the first peptide library members optionallycomprising an alkyne, azide or reporter moiety or combinations thereof;

B) contacting the first peptide library with a target or a truncatedanalogue thereof, the target or truncated analogue thereof comprising afirst binding site and optionally an alkyne, azide or reporter moiety orcombinations thereof;

C) identifying a first peptide library member with affinity for thefirst binding site and optionally modifying the first peptide librarymember to include an alkyne or azide moiety;

and optionally:

D) providing a second peptide library comprising a plurality of secondpeptide library members, the second peptide library members comprisingan azide or alkyne or both;

E) contacting the second peptide library with a composition comprisingthe target or truncated analogue thereof and the first peptide librarymember of step C;

F) forming a triazole-linked conjugate between the first peptide librarymember of step C and a second peptide library member, the second peptidelibrary member having affinity for a second binding site on the targetor truncated analogue thereof,

wherein the first peptide library, the second peptide library, or both,comprise cyclic peptides comprising:

-   -   i. a sequence region comprising amino and carboxy termini and a        variable peptide sequence of two to twenty amino acids selected        from natural and non-natural amino acids; and    -   ii. a linker region comprising a α-amino carbonyl, α-amido        carbonyl, a methionine amino acid, or combinations thereof, and        optionally comprising an alkyne, an azide, a linkage to a solid        support or a linkage to a reporter moiety or a combination        thereof, the linker region covalently linking the amino and        carboxy termini of the sequence region.

A preferred set of amino acids from which the amino acids of SEQ can beselected (Set 1) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobicside chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan(MT), 2-Methoxy Pyridylalanine (MeOPyr), and 4-Phenyl Phenylalanine(PhF) (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andβ-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro(conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 2) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF),and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andN-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic sidechain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys,Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA),N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

In some forms, SEQ can comprise t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn(SEQ ID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh,t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn(SEQ ID NO:4), t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6),t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQID NO:8), tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp(SEQ ID NO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13),tnkk(CyA) (SEQ ID NO:14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15),trrk(CyA) (SEQ ID NO:16), trrks (SEQ ID NO:17), tkrkr (SEQ ID NO:18),trkkh (SEQ ID NO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ ID NO:21), tshkr(SEQ ID NO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQ ID NO:24),tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQ ID NO:27),tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ ID NO:29), tnpks(SEQ ID NO:31), tp(CyA)k(FP), t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), or trrkr(SEQ ID NO:30).

In various other embodiments, uses of the cyclic peptides and methodsemploying the same are provided.

Also disclosed are improved methods for analyzing and assessing epitopesas potential targets for ligands and protein targeting agents.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or can be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph binding of some example peptides to CD8+ cells (SuppT1cells) and CD8− cells (Jurkat cells).

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions can be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

Incorporating non-canonical (unnatural) amino acids can greatly increasethe chemical diversity of one bead one compound libraries. Preferrednon-canonical amino acids have unique properties that differsubstantially from the canonical 20 amino acids. These new amino acidsincorporate (but are not limited to) heterocycles, halogen-substitutedarenes, carbocycles, beta amino acids, and N-methylation. Several ofthese structures are considered “privileged scaffolds” of medicinalchemistry, moieties that often appear in bioactive compounds. Thesubstitution also decreases the intrinsic metabolic liabilities ofseveral natural amino acids. The inclusion of beta amino acids increasesthe flexibility of the macrocycle, and incorporation of N-Methylatedamino acids increases the propensity of the amine bond to adopt acis-geometry.

The standard 16 amino acid set (i.e., using canonical amino acids)contains Ala, Gly, Leu, and Val (hydrophobic side chain—aliphatic); Phe,Tyr, and Trp (hydrophobic side chain—aromatic); Asn, Ser, and Thr (polarside chain—neutral); His, Lys, Arg, and Glu (polar side chain—charged);and Pro (conformational perturbation). The canonical amino acids leftout of this set are Ile, Gln, Cys, and Met.

One preferred non-canonical 16 amino acid set (i.e., includingnon-canonical amino acids) (Set 1) contains Cyclopropyl Alanine (CyA)and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP),Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), and 4-PhenylPhenylalanine (PhF) (hydrophobic side chain—aromatic); Asn, Ser, Thr(polar side chain—neutral); His, Lys, Arg, Glu (polar sidechain—charged); and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a),and Pro (conformational perturbation). The canonical amino acids leftout of this set are Ala, Ile, Leu, Val, Phe, Tyr, Trp, Asp, Gln, Cys,and Met.

Another preferred non-canonical 16 amino acid set (i.e., includingnon-canonical amino acids) (Set 2) contains Cyclopropyl Alanine (CyA)and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP),Methyl Tryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine(PhF), and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polarside chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andN-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). Thecanonical amino acids left out of this set are Ala, Ile, Leu, Val, Tyr,Trp, Asp, Gln, Cys, and Met.

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic sidechain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys,Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA),N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation)—thestructures of which can be seen above.

In general, non-canonical amino acids that can be included in thedisclosed amino acid sets can be selected from any one or combinationsof Cyclopropyl Alanine (CyA), Methyl Leucine (MeL), Methyl Valine (MeV),Allylglycine, Methyl Tryptophan (MT), Thiazolyl Alanine (Thz),2-Naphthyl Alanine (Nap), O-Methyl Tyrosine (OMY), 4-FluorophenylAlanine (FP), 4-Cyano Phenylalanine (CN-F), 4-Phenyl Phenylalanine(PhF), 4-Bromo Phenylalanine (Br-F), 4-Pyridyl Alanine (PyrA), 4-MethylPhenylalanine (Me-F), O-Phenyl Tyrosine (OPhY), β-Phenylalanine (BPhA),Dimethyl Lysine (DMK), 2-Methoxy Pyridylalanine (MeOPyr),Piperazinecarboxylic acid, N-Methyl d-valine (N-Me-v), N-Methyld-alanine (N-Me-a), Aminocyclobutyl carboxylic acid (ACBC),Aminocyclohexyl carboxylic (ACHC), α-Methyl Alanine (AMA),Morpholinecarboxylic acid, Tetrazoyl Alanine (Ttz), β-Alanine (BAla),and Azetidine carboxylic acid.

In some forms, non-canonical amino acids that can be included in thedisclosed amino acid sets can be selected from any one or combinationsof Cyclopropyl Alanine (CyA), Methyl Leucine (MeL), Methyl Valine (MeV),Methyl Tryptophan (MT), Thiazolyl Alanine (Thz), 2-Naphthyl Alanine(Nap), O-Methyl Tyrosine (OMY), 4-Fluorophenyl Alanine (FP), 4-CyanoPhenylalanine (CN-F), 4-Phenyl Phenylalanine (PhF), 4-BromoPhenylalanine (Br-F), 4-Pyridyl Alanine (PyrA), 4-Methyl Phenylalanine(Me-F), O-Phenyl Tyrosine (OPhY), β-Phenylalanine (BPhA), DimethylLysine (DMK), N-Methyl d-valine (N-Me-v), N-Methyl d-alanine (N-Me-a),Aminocyclobutyl carboxylic acid (ACBC), Aminocyclohexyl carboxylic(ACHC), α-Methyl Alanine (AMA), Tetrazoyl Alanine (Ttz), and β-Alanine(BAla).

The non-canonical amino acids generally fall in the followingcategories:

Hydrophobic side chain—aliphatic: CyA, MeL, MeV, and allylglycine;

Hydrophobic side chain—aromatic: FP, MT, MeOPyr, PhF, Nap, OMY, CN—F,Br—F, PyrA, Me-F, OPhY; and

Conformational perturbation: BPhA, N-Me-a, and N-Me-v.

In some forms, the amino acid set can include two or more amino acidsfrom each of the categories (a) hydrophobic side chain—aliphatic, (b)hydrophobic side chain—aromatic, (c) polar side chain—neutral, (d) polarside chain—charged, and (e) conformational perturbation.

In some forms, the amino acid set can include two or more amino acidsfrom each of the categories (a) hydrophobic side chain—aliphatic, (b)hydrophobic side chain—aromatic, (c) polar side chain—neutral, and (d)polar side chain—charged, and can include one or more amino acids fromthe conformational perturbation category.

In some forms, the amino acid set can comprise 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids. Insome forms, the amino acid set can consist of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

In some forms, the amino acid set can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26different non-canonical amino acids.

In some forms, the amino acid set can comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different canonicalamino acids.

In some forms, the amino acid set can comprise any combination ofcanonical amino acids and non-canonical amino acids, wherein at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofthe amino acids in the amino acid set is a non-canonical amino acid andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 of the amino acids in the amino acid set is a canonical aminoacid, and wherein the set of amino acids totals 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

In some forms, the amino acid set can consist of any combination ofcanonical amino acids and non-canonical amino acids, wherein at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ofthe amino acids in the amino acid set is a non-canonical amino acid andat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 of the amino acids in the amino acid set is a canonical aminoacid, and wherein the set of amino acids totals 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 different amino acids.

Preferred libraries can use 16-20 amino acids with a 5-mer variableregion. This generally optimizes the mix of cost, unique mass, and OBOCsynthesis (Table 3).

TABLE 3 Total Number of Number Library Mass Library Mass (g) Beads ofBeads Over- (g) per Size Synthesized Synthesized per Copy sampling Copy16⁵ 5 14300000 1048576 13.64 0.37 17⁵ 5.3125 15193750 1419857 10.70 0.5018⁵ 5.625 16087500 1889568 8.51 0.66 19⁵ 5.9375 16981250 2476099 6.860.87 20⁵ 6.25 17875000 3200000 5.59 1.12

The libraries can be synthesized in, for example, 16-20 reaction vessels(1 reaction vessel per amino acid), with 5 split-mix cycles performed togenerate 5-mer peptides. Preferred reactions can use a maximum of 0.3125g of beads per reaction vessel. As an example, Rapp Polymere's beadshave 2.86×10⁶ TentaGel S NH₂ beads per gram. Current preferred librariesuse D-amino acid library of 16⁵ diversity with each screen containing0.5 g library (>1 copy).

Also disclosed are improved methods for analyzing and assessing epitopesas potential targets for ligands and protein targeting agents. In orderto increase our understanding of what makes a candidate sequence a goodepitope for PCC targeting, we have implemented in silico methods toincrease the likelihood of generating quality hits against the targetprotein. Examples of the attributes that may lead to a quality hitinclude the flexibility or rigidity of the epitope (within the contextof the entire protein), solvent exposed surface area, overall charge,post-translational modification, and volume of the surrounding surface.

We have used molecular dynamics software to identify areas of any givenprotein that appear to be more flexible. For this type of stimulation,the crystal structure of the protein interest is solvated in water andthe energy of the system is minimized. Due to the low temperaturerequired to crystalize biomolecules, the temperature of the systemduring the simulation is slowly increase to physiological conditions.This increases the vibrations of both the surrounding water in thesimulation and the protein itself. Portions of the protein that arehighly structured will not undergo translational, rotational, orvibrational movement to the same extent as regions that are flexible.

Other software including Pymol is utilized to identify long contiguoussequences that are predicted to be solvent exposed. This increases thelikelihood that the epitopes that we target with our PCCs will translateto hits against the full-length protein.

The following is an example of the method of assessing epitopes.

Prepare Structure

Crystal Structure Preparation: The native PDB file is downloaded fromthe RCSB PDB homepage. Any non-proteinaceous material (ligand,stabilizers, crystallographic water, etc.) is extracted and removed fromthe PDB file using Pymol.

Protein Structure File: NAMD is used to generate a PSF file, whichcontains the bonding interactions necessary to run the simulation.Partial charges and bond lengths are examples of parameters that arerequired. Force field topology files are used to interpret the PDB fileand to generate the PSF file.

Water Box Preparation: The PDB and PSF files are used to solvate theprotein in a water box with at least 5 Å (typically 10 Å for productionruns) separating the protein and cell boundary.

Minimization

Minimization Methods: The protein was minimized and equilibrated usingperiodic boundary conditions employing Particle Mesh Ewaldelectrostatics. This means that the system (water box) is surrounded byadditional water boxes during equilibration. This minimizes thetendencies of surface tension effects from throwing off the calculationsif the system was equilibrated in a vacuum.

Production Run: The system was warmed to 310° K, minimized for 1000steps (sometimes as much as 10,000 steps), and equilibrated for at least100 ps (typically 1000 or 5000 ps).

The particular features, structures, or characteristics may be combinedin any suitable manner in one or more embodiments.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Aminocarbonyl” refers to a radical of the formula —C(═O)NR_(a)R_(a),where each R_(a) is independently H, alkyl or a linker moiety.

“α-amino carbonyl” refers to a radical of the formula—C(═O)CR_(b)(NR_(a)R_(a))—, where each R_(a) is independently H, alkylor a linker moiety and R_(b) is H or alkyl. In some embodiments, analpha amino carbonyl is part of a cyclic moiety (e.g., peptide) wherethe carbonyl is within the ring and the amino (NR^(a)R^(a)) isexocyclic. For example, in certain embodiments and alpha aminocarbonylis useful for Edman degradation of cyclic peptides.

“α-amido carbonyl” refers to a radical of the formula—C(═O)CR_(b)(N(C═O)R_(a)R_(a))—, where each R_(a) is independently H,alkyl or a linker moiety and R_(b) is H or alkyl. In some embodiments,an alpha amido carbonyl is part of a cyclic moiety (e.g., peptide) wherethe carbonyl is within the ring and the amido (N(C═O)R^(a)R^(a)) isexocyclic.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include, but are not limited to, arylradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl radicalsthat are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl and thelike. Unless stated otherwise specifically in the specification, anaralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo. “Haloalkyl”refers to an alkyl radical, as defined above, that is substituted by oneor more halo radicals, as defined above, e.g., trifluoromethyl,difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl radical to the rest of the molecule is through anitrogen atom in the heterocyclyl radical. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —R_(b)R_(e) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. Unless stated otherwisespecifically in the specification, a heterocyclylalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this invention,the heteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless stated otherwise specifically inthe specification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkyl” refers to a radical of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylradical as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, alkoxy, alkylamino, aminocarbonyl, α-aminocarbonyl,α-amidocarbonyl, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl) wherein at least one hydrogen atomis replaced by a bond to a non-hydrogen atoms such as, but not limitedto: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groupssuch as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atomin groups such as thiol groups, thioalkyl groups, sulfone groups,sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such asamines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines,diarylamines, N-oxides, imides, and enamines; a silicon atom in groupssuch as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilylgroups, and triarylsilyl groups; and other heteroatoms in various othergroups. “Substituted” also means any of the above groups in which one ormore hydrogen atoms are replaced by a higher-order bond (e.g., a double-or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl,carboxyl, and ester groups; and nitrogen in groups such as imines,oximes, hydrazones, and nitriles. For example, “substituted” includesany of the above groups in which one or more hydrogen atoms are replacedwith —NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. “Substituted” further means any of the above groups inwhich one or more hydrogen atoms are replaced by a bond to an amino,cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy,alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In addition, each of theforegoing substituents may also be optionally substituted with one ormore of the above substituents.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable peptides of structure (I) or (I′) beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabelledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled peptides of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e. ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled peptidescan generally be prepared by conventional techniques known to thoseskilled in the art or by processes analogous to those described in thePreparations and Examples as set out below using an appropriateisotopically-labeled reagent in place of the non-labeled reagentpreviously employed.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed peptides. Such products may resultfrom, for example, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes.

Accordingly, the invention includes compounds produced by a processcomprising administering a compound of this invention to a mammal for aperiod of time sufficient to yield a metabolic product thereof. Suchproducts are typically identified by administering a radiolabelledcompound of the invention in a detectable dose to an animal, such asrat, mouse, guinea pig, monkey, or to human, allowing sufficient timefor metabolism to occur, and isolating its conversion products from theurine, blood or other biological samples.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a peptide of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment of adisease or condition in the mammal, preferably a human. The amount of acompound of the invention which constitutes a “therapeutically effectiveamount” will vary depending on the compound, the condition and itsseverity, the manner of administration, and the age of the mammal to betreated, but can be determined routinely by one of ordinary skill in theart having regard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds (peptides) of the invention, or their pharmaceuticallyacceptable salts may contain one or more asymmetric centers and may thusgive rise to enantiomers, diastereomers, and other stereoisomeric formsthat may be defined, in terms of absolute stereochemistry, as (R)- or(S)- or, as (D)- or (L) for amino acids. The present invention is meantto include all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

The term “capture agent” as used herein refers to a protein-catalyzedcapture (PCC) agent that comprises one or more target-binding moietiesand which specifically binds to a target protein via thosetarget-binding moieties. Each target-binding moiety exhibits bindingaffinity for the target protein, either individually or in combinationwith other target-binding moieties. In certain embodiments, eachtarget-binding moiety binds to the target protein via one or morenon-covalent interactions, including for example hydrogen bonds,hydrophobic interactions, and van der Waals interactions. A captureagent may comprise one or more organic molecules, including for examplepolypeptides, peptides, polynucleotides, and other non-polymericmolecules.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to an amino acid sequence comprising apolymer of amino acid residues. The terms apply to amino acid polymersin which one or more amino acid residues is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids, andisomers thereof. Naturally occurring amino acids are those encoded bythe genetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, carboxyglutamate, O-phosphoserine, and isomersthereof. The term “amino acid analogs” refers to compounds that have thesame basic chemical structure as a naturally occurring amino acid, i.e.,a carbon that is bound to a hydrogen, a carboxyl group, an amino group,and an R group, e.g., homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. The term “aminoacid mimetics” refers to chemical compounds that have a structure thatis different from the general chemical structure of an amino acid, butthat functions in a manner similar to a naturally occurring amino acid.Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The term “non-natural amino acid” as used herein refers to an amino acidthat is different from the twenty naturally occurring amino acids(alanine, arginine, glycine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, serine, threonine, histidine, lysine,methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan,phenylalanine) in its side chain functionality. The non-natural aminoacid can be a close analog of one of the twenty natural amino acids, orit can introduce a completely new functionality and chemistry, as longas the hydrophobicity of the non-natural amino acid is either equivalentto or greater than that of the natural amino acid. The non-natural aminoacid can either replace an existing amino acid in a protein(substitution), or be an addition to the wild type sequence (insertion).The incorporation of non-natural amino acids can be accomplished byknown chemical methods including solid-phase peptide synthesis or nativechemical ligation, or by biological methods.

The terms “specific binding,” “selective binding,” “selectively binds,”or “specifically binds” as used herein refer to non-random binding of abinding agent (target binding compound) such as a capture agent to anepitope on a predetermined antigen. Binding agents (e.g., peptides)which specifically bind to a target are also referred to as havingaffinity for the target, or a binding site thereon. Typically, thebinding agent binds with an affinity (KD) of approximately less than10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M oreven lower.

The term “KD” as used herein refers to the dissociation equilibriumconstant of a particular interaction between a binding agent such as acapture agent and its antigen. Typically, the binding agents of theinvention bind to a target (e.g., AKT) with a dissociation equilibriumconstant (KD) of less than approximately 10⁻⁷ M, such as less thanapproximately 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ M or even lower, for example, asdetermined using surface plasmon resonance (SPR) technology in a Biacoreinstrument using the antigen as the ligand and the capture agent as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a KD that is at least ten-fold lower, such as at least100 fold lower, for instance at least 1,000 fold lower, such as at least10,000 fold lower, for instance at least 100,000 fold lower than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen. The amountwith which the affinity is lower is dependent on the KD of the captureagent, so that when the KD of the capture agent very low (that is, thecapture agent is highly specific), then the amount with which theaffinity for the antigen is lower than the affinity for a non-specificantigen may be at least 10,000 fold.

The term “kd” (sec′) as used herein refers to the dissociation rateconstant of a particular binding agent-antigen interaction. Said valueis also referred to as the koff value.

The term “ka” (M-‘xsec’) as used herein refers to the association rateconstant of a particular binding agent-antigen interaction.

The term “KD” (M) as used herein refers to the dissociation equilibriumconstant of a particular binding agent-antigen interaction.

The term “KA” (M-′) as used herein refers to the association equilibriumconstant of a particular binding agent-antigen interaction and isobtained by dividing the ka by the kd.

The term “condition” as used herein refers generally to a disease,event, or a change in health status. A change in health status may beassociated with a particular disease or event, in which case the changemay occur simultaneously with or in advance of the disease or event. Inthose cases where the change in health status occurs in advance of adisease or event, the change in health status may serve as a predictorof the disease or event. For example, a change in health status may bean alteration in the expression level of a particular gene associatedwith a disease or event. Alternatively, a change in health status maynot be associated with a particular disease or event.

The term “antibody” as used herein refers to a protein of the kind thatis produced by activated B cells after stimulation by an antigen and canbind specifically to the antigen promoting an immune response inbiological systems. Full antibodies typically consist of four subunitsincluding two heavy chains and two light chains. The term antibodyincludes natural and synthetic antibodies, including but not limited tomonoclonal antibodies, polyclonal antibodies or fragments thereof.Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and thelike. Exemplary fragments include Fab, Fv, Fab′, F(ab′)2 and the like. Amonoclonal antibody is an antibody that specifically binds to and isthereby defined as complementary to a single particular spatial andpolar organization of another biomolecule which is termed an “epitope.”In some forms, monoclonal antibodies can also have the same structure. Apolyclonal antibody refers to a mixture of different monoclonalantibodies. In some forms, polyclonal antibodies can be a mixture ofmonoclonal antibodies where at least two of the monoclonal antibodiesbinding to a different antigenic epitope. The different antigenicepitopes can be on the same target, different targets, or a combination.Antibodies can be prepared by techniques that are well known in the art,such as immunization of a host and collection of sera (polyclonal) or bypreparing continuous hybridoma cell lines and collecting the secretedprotein (monoclonal).

The term “stable” as used herein with regard to the disclosed peptidesor pharmaceutical formulation thereof means that the agent orformulation maintains structural and functional integrity for asufficient period of time to be useful in the methods described herein.

The term “synthetic” as used herein with regard to the disclosedpeptides means that the capture agent has been generated by chemicalrather than biological means.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” or “substantially identical” ofpolynucleotide sequences means that a polynucleotide comprises asequence that has between 50-100% sequence identity, preferably at least50% sequence identity, preferably at least 60% sequence identity,preferably at least 70%, more preferably at least 80%, more preferablyat least 90% and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of between 55-100%, preferably at least55%, preferably at least 60%, more preferably at least 70%, 80%, 90% andmost preferably at least 95%.

It is to be understood that the disclosed method and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

In situ click chemistry (J. Am. Chem. Soc. 126:12809 (2004); Angew.CHem. Int. Ed. Engl. 44:116 (2004); Angew. Chem. Int. Ed. Engl. 45:1435(2006)) is a technique in which a small molecule enzymatic inhibitor isseparated into two moieties, each of which is then expanded into a smalllibrary—one containing acetylene functionalities, and the othercontaining azide groups. The enzyme itself then assembles the ‘best fit’inhibitor from these library components by selectively promoting1,3-dipolar cycloaddition between the acetylene and azide groups to forma triazole linkage (the ‘click’ reaction). The enzyme promotes the clickreaction only between those library components that bind to the proteinin the right orientation. The resultant inhibitor can exhibit farsuperior affinity characteristics relative to the initial inhibitor thatformed the basis of the two libraries (Proc. Natl. Acad. Sci. USA97:9367 (1981); J. Comput. Aided. Mol. Des. 16:741 (2002)).

Sequential in situ click chemistry extends the in situ click chemistryconcept to enable the discovery of multiligand capture agents. Thisprocess was used previously to produce a triligand capture agent againstthe model protein carbonic anhydrase II (CAII) (Angew. Chem. Int. Ed.Engl. 48:4944 (2009)). Sequential in situ click chemistry has severaladvantages. First, structural information about the protein target isreplaced by the ability to sample a very large chemical space toidentify the ligand components of the capture agent. For example, aninitial ligand may be identified by screening the protein against alarge (>10⁶ element) one-bead-one-compound (OBOC) (Nature 354:83 (1991))peptide library, where the peptides themselves may be comprised ofnatural, non-natural, and/or artificial amino acids. The resultantanchor ligand is then utilized in an in situ click screen, again using alarge OBOC library, to identify a biligand binder. A second advantage isthat the process can be repeated, so that the biligand is used as ananchor to identify a triligand, and so forth. The final capture agentcan then be scaled up using relatively simple and largely automatedchemistries, and it can be developed with a label, such as a biotingroup, as an intrinsic part of its structure. This approach permits theexploration of branched, cyclic, and linear capture agent architectures.While many strategies for protein-directed multiligand assembly havebeen described (Science 274:1531 (1996); Proc. Natl. Acad. Sci. USA97:9367 (2000)), most require detailed structural information on thetarget to guide the screening strategy, and most (such as the originalin situ click approach), are optimized for low-diversity small moleculelibraries.

In some forms, the peptide is a cyclic peptide having the followingstructure (I):

or a salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹ and L² are each individually optionally substituted linker moieties,each linker moiety optionally comprising a linkage to a solid support, alinkage to a reporter moiety, a linkage to a peptide ligand, a linkageto an alkyne or azide moiety or combinations thereof;

G is a triazole, a carbon-carbon double bond or an amide;

M is methionine;

R is H, -L³-A or —C(═O)-L³-A, where L³ is a linker moiety and A is analkyne, azide or a bond to a peptide ligand;

R¹ is H or C₁-C₆alkyl;

Y¹ and Y² are each individually 0 or 1; and

SEQ is an amino acid sequence comprising from 2 to 20 amino acidsselected from natural and non-natural amino acids.

A preferred set of amino acids from which the amino acids of SEQ can beselected (Set 1) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobicside chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan(MT), 2-Methoxy Pyridylalanine (MeOPyr), and 4-Phenyl Phenylalanine(PhF) (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andβ-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro(conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 2) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF),and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andN-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic sidechain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys,Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA),N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

In some embodiments, G is a triazole. Such triazoles may be derived byreaction of an alkyne and azide on a precursor acyclic peptide.

In other embodiments, G is a carbon-carbon double bond. In someembodiments, these peptides are obtained by reactions of twocarbon-carbon double bonds (alkenes) present in an acyclic precursor.Such reactions can be carried out using Grubbs metathesis chemistry,which is well-known to those of skill in the art.

In various other embodiments, L¹, L², or both, comprise one or moresubstituents selected from alkyl, alkyne, azide and aminocarbonyl. Insome other embodiments of any of the foregoing, L¹, L², or both,comprise a linkage selected from a linkage to a solid support, a linkageto a reporter moiety and a linkage to a peptide ligand. In some specificembodiments of the foregoing, L¹ and L² are alkylene.

In some other embodiments of the foregoing, the cyclic peptide has oneof the following structures (Ia) or (Ib):

wherein:

R³ is H, a linkage to a solid support, a linkage to a reporter moiety, alinkage to a peptide ligand, a linkage to an alkyne or azide moiety orcombinations thereof; and

x and y are each independently an integer from 1 to 8.

In some embodiments of the compounds of structure (Ia) and (Ib), x is 1.In other embodiments, x is 2. In more embodiments, x is 3. In stillother embodiments, x is 4. In other embodiments, x is 5. In some otherembodiments, x is 6. In yet more embodiments, x is 7. In otherembodiments, x is 8.

In some embodiments of the compounds of structure (Ia) and (Ib), y is 1.In other embodiments, y is 2. In more embodiments, y is 3. In stillother embodiments, y is 4. In other embodiments, y is 5. In some otherembodiments, y is 6. In yet more embodiments, y is 7. In otherembodiments, y is 8.

In other embodiments, R is H or —C(═O)-L³-A, where L³ is a linker moietyand A is a bond to a peptide ligand or an alkyne. In some of theseembodiments, A is an alkyne. In other embodiments the cyclic peptide isbiligand binding agent, and A is a bond to a peptide ligand, for examplea linear peptide ligand or a cyclic peptide ligand. In furtherembodiments, the peptide ligand further comprises a second peptideligand, and the cyclic peptide is this a tri-ligand binding agent.

The structure of the “linker moieties” (e.g., linker moieties toreporter moieties or further peptides, etc.) are not particularlylimited. For example, in certain embodiments, linkers comprisingethylene glycol of various lengths (e.g., 1-10 glycol repeating units,e.g., about 5-7). Ethylene diamine linkers may also be employed alone orin combination with other moieties (e.g., ethylene glycol). Linkermoieties comprising triazole (e.g., resulting from reaction of an alkyneand azide) are also useful in various embodiments.

In some of the foregoing embodiments, y¹ and y² are each 0.

In even more embodiments, the cyclic peptide has one of the followingstructures:

For example, in some of any of the foregoing embodiments SEQ comprisesfrom 2 to 9 amino acids. In other embodiments, SEQ comprises from 5 to 7amino acids.

In certain embodiments, SEQ comprise natural amino acids. In otherembodiments, SEQ comprises non-natural amino acids. In still moreembodiments, SEQ comprises natural and non-natural amino acids.

In some more specific embodiments any of the foregoing embodiments, theamino acids are selected from D and L stereoisomers of Ala, Gly, Leu,Ile, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Gln, Ser, Thr, Tyr andPro. In some more specific embodiments any of the foregoing embodiments,the amino acids are selected from D and L stereoisomers of Ala, Gly,Leu, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Ser, Thr, Tyr and Pro.In some more specific embodiments any of the foregoing embodiments, theamino acids are selected from CyA, Gly, FP, MT, MeOPyr, PhF, Asn, Ser,Thr, His, Lys, Arg, Glu, BPhA, N-Me-a, and Pro (Set 1). In some morespecific embodiments any of the foregoing embodiments, the amino acidsare selected from CyA, Gly, FP, MT, Thz, PhF, Phe, Asn, Ser, Thr, His,Lys, Arg, Glu, N-Me-a, and Pro (Set 2). In some more specificembodiments any of the foregoing embodiments, the amino acids areselected from CyA, Gly, FP, MT, MeOPyr, Thz, PhF, Phe, Asn, Ser, Thr,His, Lys, Arg, Glu, BPhA, N-Me-a, and Pro (Set 3).

The amino acids in SEQ are selected to have affinity for the desiredtarget, including allosteric binding sites such as protein epitopes.

Compositions comprising any of the foregoing cyclic peptides and apharmaceutically acceptable carrier are also provided in variousembodiments. In other embodiments, a library comprising a plurality ofthe forgoing cyclic peptides is provided.

-   -   In certain embodiments, the cyclic peptides (also referred to        herein as capture agents or binding agents) provided herein have        a shelf-life of greater than six months, meaning that they are        stable in storage for greater than six months. In certain of        these embodiments, the capture agents have a shelf-life of one        year or greater, two years or greater, or more than three years.        In certain of these embodiments, the capture agents are stored        as a lyophilized powder. In certain embodiments, the capture        agents provided herein have a longer shelf-life than a biologic        binding to the same target protein.        In certain embodiments, the capture agents provided herein are        stable at temperatures ranging from about −80° C. to about        120° C. In certain of these embodiments, the capture agents are        stable within a temperature range of −80° C. to −40° C.; −40° C.        to −20° C.; −20° C. to 0° C.; 0° C. to 20° C.; 20° C. to 40° C.;        40° C. to 60° C.; 60° C. to 80° C.; and/or 80° C. to 120° C. In        certain embodiments, the capture agents provided herein are        stable across a wider range of temperatures than a biologic        binding to the same target protein, and/or remain stable at a        specific temperature for a longer time period than a biologic        binding to the same target protein.

In certain embodiments, the pH of a capture agent provided herein is inthe range of about 3.0 to about 12.0. In certain of these embodiments,the pH of the capture agent is in the range of about 5.0 to about 9.0.The pH of a capture agent may be adjusted to a physiologicallycompatible range using methods known in the art. For example, in certainembodiments the pH of the capture agent may be adjusted to the range ofabout 6.5 to about 8.5.

In certain embodiments, the capture agents provided herein are stable inblood serum for more than 12 hours. In certain of these embodiments, thecapture agents are stable in blood serum for more than 18 hours, morethan 24 hours, more than 36 hours, more than 48 hours, or more than 96hours. In certain embodiments, the capture agents provided herein arestable for a longer period of time in blood serum than a biologicbinding to the same target protein.

In certain embodiments, the capture agents provided herein may compriseone or more detection labels (reporter group), including for examplebiotin, copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid(copper-DOTA), desferrioxamine B (DFO), a ligand for radiolabeling with⁶⁸Ga, or other radiolabeled products that may include gamma emitters,proton emitters, positron emitters, tritium, or covered tags detectableby other methods (i.e., gadolinium) among others.

In certain embodiments, the capture agents provided herein comprise oneor more detectable labels. In certain of these embodiments, the label iscopper-DOTA. In other embodiments, the detectable label is selected from⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴I, ⁸⁶Y, ^(94m)Tc,^(110m)In, ¹¹C and ⁷⁶Br. In other embodiments, the detectable label isselected from ¹²³I, ¹³¹I, ⁶⁷Ga, ¹¹¹In and ^(99m)Tc. In otherembodiments, the label is a fluorescent label.

In some other embodiments, the cyclic peptide comprises a linkage to areporter moiety, the reporter moiety selected from polyethylene glycol(PEG), biotin, thiol and fluorophores. For example, in some embodimentsthe fluorophores are selected from FAM, FITC, Cy5, TRITC, TAMRA.

Table 1 provides reporter moieties useful in various differentapplications of the cyclic peptides. Other useful reporter moieties canbe derived by one of skill in the art.

TABLE 1 Reporter Moieties Application Reporter ELISA: microtiter plateBiotin ELISA: lateral flow test Biotin Immunoprecipitation (and otherBiotin, thiol bead-based assays) Dot blot Biotin Cell-based assayBiotin, fluorophore IHC Biotin, fluorophore In vivo imaging: PETRadioisotopes including ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, ⁸⁹Zr, ¹²⁴I In vivo imaging:SPECT Radioisotopes including ¹¹¹In, ⁹⁰Y, ^(99m)Tc In vivo imaging: MRGd³⁺

In certain embodiments, the capture agents provided herein may bemodified to obtain a desired chemical or biological activity. Examplesof desired chemical or biological activities include, withoutlimitation, improved solubility, stability, bioavailability,detectability, or reactivity. Examples of specific modifications thatmay be introduced to a capture agent include, but are not limited to,cyclizing the capture agent through formation of a disulfide bond;modifying the capture agent with other functional groups or molecules.Similarly, a capture agent may be synthesized to bind to non-canonicalor non-biological epitopes on proteins, thereby increasing theirversatility. In certain embodiments, the capture agent may be modifiedby modifying the synthesis blocks of the target-binding moieties beforethe coupling reaction.

Provided herein in certain embodiments are pharmaceutical formulationscomprising one or more of the capture agents provided herein. In certainembodiments, these pharmaceutical formulations comprise one or morepharmaceutically acceptable carriers, excipients, or diluents. Thesecarriers, excipients, or diluents may be selected based on the intendeduse and/or route of administration of the formulation.

Provided herein in certain embodiments are kits comprising one or moreof the capture agents disclosed herein. In certain embodiments, the kitsprovided herein may further comprise instructions for suitableoperational parameters in the form of a label or a separate insert. Forexample, the kit may have standard instructions informing a consumer/kituser how to wash the probe after a sample of plasma or other tissuesample is contacted on the probe.

It is understood that any embodiment of the peptides, as set forthabove, and any specific substituent set forth herein for a R, R¹, L¹,L², G, M, Y¹ Y² or SEQ group in the peptides, as set forth above, may beindependently combined with other embodiments and/or substituents of thepeptides to form embodiments of the inventions not specifically setforth above. In addition, in the event that a list of substituents islisted for any particular variable in a particular embodiment and/orclaim, it is understood that each individual substituent may be deletedfrom the particular embodiment and/or claim and that the remaining listof substituents will be considered to be within the scope of theinvention.

For the purposes of administration, the peptides of the presentinvention may be administered as a raw chemical or may be formulated aspharmaceutical compositions. Pharmaceutical compositions of the presentinvention comprise a peptide of structure (I) and a pharmaceuticallyacceptable carrier, diluent or excipient. The peptide of structure (I)is present in the composition in an amount which is effective to treat aparticular disease or condition of interest—that is, and preferably withacceptable toxicity to the patient. Activity of compounds of thepeptides can be determined by one skilled in the art, for example, asdescribed in the Examples. Appropriate concentrations and dosages can bereadily determined by one skilled in the art.

Administration of the compounds of the invention, or theirpharmaceutically acceptable salts, in pure form or in an appropriatepharmaceutical composition, can be carried out via any of the acceptedmodes of administration of agents for serving similar utilities. Thepharmaceutical compositions of the invention can be prepared bycombining a compound of the invention with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical compositions of the invention areformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a subject or patient take theform of one or more dosage units, where for example, a tablet may be asingle dosage unit, and a container of a compound of the invention inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy andScience, 2000). The composition to be administered will, in any event,contain a therapeutically effective amount of a compound of theinvention, or a pharmaceutically acceptable salt thereof, for treatmentof a disease or condition of interest in accordance with the teachingsof this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, aprotein or a liposome.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining a compound of the invention with sterile,distilled water so as to form a solution. A surfactant may be added tofacilitate the formation of a homogeneous solution or suspension.Surfactants are compounds that non-covalently interact with the compoundof the invention so as to facilitate dissolution or homogeneoussuspension of the compound in the aqueous delivery system.

The compounds of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific compound employed; the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, anddiet of the patient; the mode and time of administration; the rate ofexcretion; the drug combination; the severity of the particular disorderor condition; and the subject undergoing therapy.

Compounds of the invention, or pharmaceutically acceptable derivativesthereof, may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy includes administration of a single pharmaceuticaldosage formulation which contains a compound of the invention and one ormore additional active agents, as well as administration of the compoundof the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a compound of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The peptides of the invention can be prepared by procedures known tothose of skill in the art. For example, the peptides can be preparedusing standard solid-phase peptide synthesis techniques, andmodifications thereof. Modified amino acids may be employed toincorporate amino acids comprising alkyne and/or azide moieties and/oralkene moieties useful for cyclization. Methods for cyclizing thepeptides using azide/alkyne chemistry and Grubbs metathesis chemistryare well-known in the art. Such methods are described in more detail inthe examples.

It is understood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, otherpeptides not specifically illustrated in the examples below by using theappropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Methods for Use of the Peptides

In various embodiments, the present invention provides methods foridentification of cyclic peptides which are useful as binding agents forvarious targets. In general, the methods employ cyclic peptides, such asany of the cyclic peptides described herein above, in methods foridentification of mono-, bi- and/or tri-ligand binding agents. Higherorder binding agents (tetra, penta, and the like) are also within thescope of the present invention.

In general, the present invention includes any methods employing thecyclic peptides described herein. Accordingly, in one embodiment amethod for identifying a target binding compound (e.g., a proteincapture agent) is provided, the method comprising

A). providing a peptide library comprising a plurality of cyclicpeptides comprising:

i. a sequence region comprising amino and carboxy termini and a variablepeptide sequence of two to twenty amino acids selected from natural andnon-natural amino acids; and

ii. a linker region comprising a α-amino carbonyl, α-amido carbonyl, amethionine amino acid, or combinations thereof, and optionallycomprising an alkyne, an azide, a linkage to a solid support or alinkage to a reporter moiety or a combination thereof, the linker regioncovalently linking the amino and carboxy termini of the sequence region.

B) contacting the peptide library with a target or a truncated analoguethereof, the target or truncated analogue thereof comprising a bindingsite and optionally an alkyne, azide or reporter moiety or combinationsthereof;

C) identifying a peptide library member with affinity for the bindingsite

In further embodiments, a method for identifying a target bindingcompound (e.g., a protein capture agent) is provided, the methodcomprising:

A) providing a first peptide library comprising a plurality of firstpeptide library members, the first peptide library members optionallycomprising an alkyne, azide or reporter moiety or combinations thereof;

B) contacting the first peptide library with a target or a truncatedanalogue thereof, the target or truncated analogue thereof comprising afirst binding site and optionally an alkyne, azide or reporter moiety orcombinations thereof;

C) identifying a first peptide library member with affinity for thefirst binding site and optionally modifying the first peptide librarymember to include an alkyne or azide moiety;

and optionally:

D) providing a second peptide library comprising a plurality of secondpeptide library members, the second peptide library members comprisingan azide or alkyne or both;

E) contacting the second peptide library with a composition comprisingthe target or truncated analogue thereof and the first peptide librarymember of step C;

F) forming a triazole-linked conjugate between the first peptide librarymember of step C and a second peptide library member, the second peptidelibrary member having affinity for a second binding site on the targetor truncated analogue thereof,

wherein the first peptide library, the second peptide library, or both,comprise cyclic peptides comprising:

i. a sequence region comprising amino and carboxy termini and a variablepeptide sequence of two to twenty amino acids selected from natural andnon-natural amino acids; and

ii. a linker region comprising a α-amino carbonyl, α-amido carbonyl, amethionine amino acid, or combinations thereof, and optionallycomprising an alkyne, an azide, a linkage to a solid support or alinkage to a reporter moiety or a combination thereof, the linker regioncovalently linking the amino and carboxy termini of the sequence region.

A preferred set of amino acids from which the amino acids of SEQ can beselected contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic sidechain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT),2-Methoxy Pyridylalanine (MeOPyr), and 4-Phenyl Phenylalanine (PhF)(hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andβ-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro(conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 2) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF),and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar sidechain—neutral); His, Lys, Arg, Glu (polar side chain—charged); andN-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

Another preferred set of amino acids from which the amino acids of SEQcan be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly(hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic sidechain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys,Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA),N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation).

In some forms, SEQ can comprise t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn(SEQ ID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh,t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn(SEQ ID NO:4), t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6),t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQID NO:8), tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp(SEQ ID NO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13),tnkk(CyA) (SEQ ID NO:14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15),trrk(CyA) (SEQ ID NO:16), trrks (SEQ ID NO:17), tkrkr (SEQ ID NO:18),trkkh (SEQ ID NO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ ID NO:21), tshkr(SEQ ID NO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQ ID NO:24),tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQ ID NO:27),tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ ID NO:29), tnpks(SEQ ID NO:31), tp(CyA)k(FP), t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), or trrkr(SEQ ID NO:30).

For purposes of clarity, it should be noted that steps D-F are optionaland the above described method is not limited to methods which requireconjugation of a second peptide. It is understood that when steps D-Fare not performed, the first library comprises the cyclic peptide;however when steps D-F are performed the cyclic peptides may be a partof either the first, second or both libraries. It should also beemphasized that the methods are not limited to identification of mono orbi-ligand binding agents, and the methods described herein can beextrapolated to identification of tertiary, ternary and higher bindingagents (e.g., by performing steps analogous to steps D-F). In general,any of the cyclic peptides described herein above may be employed in theabove methods. Specific embodiments of the peptides useful in the someembodiments of the methods are illustrated herein.

In certain embodiments of the method, the linker region comprises aα-amino carbonyl group bound to the amino terminus of the peptidesequence. In some of these embodiments, the method further comprisesdetermining the peptide sequence of one or more of the cyclic peptidesby Edman degradation.

In other embodiments, the linker region comprises a methionine aminoacid bound to the amino terminus of the variable peptide sequence. Insome of these embodiments, the method further comprises treating one ormore of the cyclic peptides with CNBr and determining the sequencethereof by mass spectrometry.

In other embodiments of the foregoing methods, the linker regioncomprises an alkyne or azide, the target or a truncated analogue thereofcomprises an alkyne or azide and identifying the first peptide librarymember with affinity for the first binding site comprises identifyingfirst peptide library members which form a triazole linkage with thetarget or a truncated analogue thereof.

In some embodiments, the first peptide library is contacted with atruncated analogue of the target.

In other aspects, the method further comprises modifying the triazolelinked conjugate to contain a triazole or alkyne and contacting themodified conjugate with the target or truncated analogue thereof and athird peptide library, the third peptide library comprising a pluralityof third peptide library members, each third peptide library membercomprising an azide or alkyne.

In still other embodiments, the method further comprises forming atriazole linkage between the modified conjugate and a member of thethird peptide library, the third peptide library member having affinityfor a third binding site on the target or truncated analogue thereof.

In some embodiments, the first binding site is an epitope. In otherembodiments, the second binding site is an epitope. In some moreembodiments, the third binding site is an epitope.

In various different embodiments, the linker region comprises acarbon-carbon double bond or a triazole.

In some embodiments the cyclic peptides have the following structure(I′):

or a salt, tautomer or stereoisomer thereof, wherein:

L¹ and L² are each individually optionally substituted linker moieties,each linker moiety optionally comprising a linkage to a solid support, alinkage to a reporter moiety, a linkage to an alkyne or azide moiety ora linkage to a peptide ligand;

G is a triazole a carbon-carbon double bond or an amide;

M is methionine;

R is H, -L³-A or —C(═O)-L³-A, where L³ is a linker moiety and A is analkyne or azide;

R¹ is H or C₁-C₆alkyl;

Y¹ and Y² are each individually 0 or 1; and

SEQ is the variable peptide sequence.

In some embodiments, Y¹ and Y² are each 0.

For example, in some of any of the foregoing embodiments SEQ comprisesfrom 2 to 9 amino acids. In other embodiments, SEQ comprises from 5 to 7amino acids.

In certain embodiments, SEQ comprise natural amino acids. In otherembodiments, SEQ comprises non-natural amino acids. In still moreembodiments, SEQ comprises natural and non-natural amino acids.

In some more specific embodiments any of the foregoing embodiments, theamino acids are selected from D and L stereoisomers of Ala, Gly, Leu,Ile, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Gln, Ser, Thr, Tyr andPro. In some more specific embodiments any of the foregoing embodiments,the amino acids are selected from D and L stereoisomers of Ala, Gly,Leu, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Ser, Thr, Tyr and Pro.In some more specific embodiments any of the foregoing embodiments, theamino acids are selected from CyA, Gly, FP, MT, MeOPyr, PhF, Asn, Ser,Thr, His, Lys, Arg, Glu, BPhA, N-Me-a, and Pro.

The amino acids in SEQ are selected to have affinity for the desiredtarget, including allosteric binding sites such as protein epitopes.

In other embodiments, the cyclic peptides have the following structure(I′a):

wherein:

L^(1a) is a linker moiety optionally substituted with one or moresubstituent selected from a linkage to an alkyne or azide moiety, alinkage to a solid support and a linkage to a reporter moiety.

In some embodiments, L^(1a) and L² are each independently optionallysubstituted alkylene.

In other embodiments, the cyclic peptides have the following structure(I′b):

wherein:

R³ is H, a linkage to a solid support, a linkage to an alkyne or azidemoiety or a linkage to a reporter moiety;

R⁴ is H or C₁-C₆ alkyl; and

x and y are each independently integers from 1 to 8.

In certain of the forgoing embodiments, G is a triazole. In otherembodiments, G is a carbon-carbon double bond.

In some more specific embodiments, the cyclic peptides have one of thefollowing structures:

In still other embodiments, the first peptide library comprises thecyclic peptides, and the second peptide library comprises linear peptidemembers.

In different embodiments, the second peptide library comprises thecyclic peptides, and the first peptide library comprises linear peptidemembers. In more embodiments, the first and second peptide librarycomprises the cyclic peptides.

In some embodiments, the target is a protein, for example a proteinepitope. In some embodiments, the protein is an enzyme or cell surfaceprotein.

Provided herein in certain embodiments are methods of using the captureagents disclosed herein to identify, detect, quantify, and/or separatetarget proteins in a biological sample. The capture agents disclosedherein can serve as a drop-in replacement for monoclonal antibodies inbiochemical assays. Therefore, in certain embodiments the methodsprovided herein utilize an immunoassay, with the capture agent replacingan antibody or its equivalent. In certain embodiments, the immunoassaymay be a Western blot, pull-down assay, dot blot, or ELISA.

A biological sample for use in the methods provided herein may beselected from the group consisting of organs, tissue, bodily fluids, andcells. Where the biological sample is a bodily fluid, the fluid may beselected from the group consisting of blood, blood serum, plasma, urine,sputum, saliva, stool, spinal fluid, cerebral spinal fluid, lymph fluid,skin secretions, respiratory secretions, intestinal secretions,genitourinary tract secretions, tears, and milk.

Provided herein in certain embodiments are methods of identifying,detecting, quantifying, and/or localizing a target protein in vivo. Incertain of these embodiments, the capture agents may be used as animaging agent. In these embodiments, the capture agents may comprise oneor more detection labels as discussed above.

Provided herein in certain embodiments are methods of using the captureagents disclosed herein to inhibit a target protein activity. In certainof these embodiments, the capture agents inhibit target protein activityby blocking binding of the target protein to its native substrate.

Provided herein in certain embodiments are methods of using the captureagents disclosed herein to diagnose and/or classify (e.g., stage) acondition associated with increased target protein expression and/oractivity. In certain embodiments, these methods comprise (a) obtaining abiological sample from a subject; (b) measuring the presence or absenceof target protein in the sample with the capture agent; (c) comparingthe levels of target protein to a predetermined control range for targetprotein; and (d) diagnosing a condition associated with increased targetprotein expression based on the difference between target protein levelsin the biological sample and the predetermined control.

In certain embodiments of the diagnosis and/or classification methodsprovided herein, the capture agents may be used to diagnose a change inhealth status in a subject, wherein the change in health status is apredictor of a disease or event. In certain of these embodiments, themethods may be utilized to predict the development of a disease or eventin a subject who does not yet exhibit any symptoms of the disease orevent. In certain embodiments, the change in health status may be anincrease in target protein levels.

Provided herein in certain embodiments are methods of treating acondition associated with increased target protein expression and/oractivity in a subject in need thereof by administering a therapeuticallyeffective amount of one or more of the capture agents or pharmaceuticalformulations disclosed herein. In certain of these embodiments, thecapture agent(s) may be linked to one or more additional therapeuticagents, including for example a chemotherapeutic agent. In preferredembodiments, the capture agent is administered as a pharmaceuticalcomposition.

A capture agent or pharmaceutical formulation may be administered to apatient in need of treatment via any suitable route. Routes ofadministration may include, for example, parenteral administration(including subcutaneous, intramuscular, intravenous, by means of, forexample a drip patch). Further suitable routes of administration include(but are not limited to) oral, rectal, nasal, topical (including buccaland sublingual), infusion, vaginal, intradermal, intraperitoneally,intracranially, intrathecal and epidural administration oradministration via oral or nasal inhalation, by means of, for example anebulizer or inhaler, or by an implant.

A capture agent or pharmaceutical formulation may also be administeredvia microspheres, liposomes, other microparticulate delivery systems orsustained release formulations placed in certain tissues includingblood. Suitable examples of sustained release carriers includesemi-permeable polymer matrices in the form of shared articles, e.g.,suppositories or microcapsules. Examples of the techniques and protocolsmentioned above and other techniques and protocols which may be used inaccordance with the invention can be found in Remington's PharmaceuticalSciences, 18th edition, Gennaro, A. R., Lippincott Williams & Wilkins;20th edition (Dec. 15, 2000) ISBN 0-912734-04-3 and PharmaceuticalDosage Forms and Drug Delivery Systems; Ansel, N. C. et al. 7th EditionISBN 0-683305-72-7, the entire disclosures of which are hereinincorporated by reference.

Provided herein in certain embodiments is the use of the capture agentsdisclosed herein in the preparation of a medicament for treating acondition associated with increased target protein expression and/oractivity.

In other more specific embodiments, the invention is directed to amethod of detecting a target protein in a sample, the method comprisingreplacing an antibody or its equivalent in a cell-based or animmunoassay with any of the foregoing cyclic peptides. In someembodiments, the immunoassay is a Western blot, a pull-down assay, a dotblot or an ELISA.

In other embodiments, a method for inhibiting activity of a protein in asubject is provided, the method comprising administering an effectiveamount of any of the foregoing cyclic peptides to a subject in needthereof.

Other embodiments are directed to a method of purifying a target, themethod comprising immobilizing any of the foregoing cyclic peptides in acolumn based format, contacting the column with a matrix containing thetarget, washing the column, and eluting the target.

Methods for imaging are also provided. For example, in one embodimentthe invention provides a method of imaging in vivo target expression,the method comprising:

a) providing any of the foregoing cyclic peptides, wherein SEQ is apeptide sequence having affinity for a location on or near a targetexpressing site in a subject, and modifying the cyclic peptide toinclude a small-molecule positron-emission-tomography ligand (PETligand);

b) administering the cyclic peptide of step a) to the subject;

c) measuring the positron emission from the PET ligand at a first time;

d) measuring the positron emission from the PET ligand at a second time;and

e) comparing the positron emission from the PET ligand at the first andsecond times.

In certain embodiments of the foregoing, the PET ligand comprises amoiety selected from ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ⁶⁸Ga NOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga,⁸⁹Z, ¹²⁴I, ⁸⁶Y, ^(94m)Tc, ^(110m)In, ¹¹C and ⁷⁶Br.

In other embodiments, the imaging method is a method of imaging in vivotarget expression, the method comprising:

a) providing any of the foregoing cyclic peptides, wherein SEQ is apeptide sequence having affinity for a location on or near a targetexpressing site in a subject, and modifying the cyclic peptide toinclude a small-molecule single-photon-emission-computed-tomographyligand (SPECT ligand);

b) administering the cyclic peptide of step a) to the subject;

c) measuring the photon emission from the SPECT ligand at a first time;

d) measuring the photon emission from the SPECT ligand at a second time;and

e) comparing the photon emission from the SPECT ligand at the first andsecond times.

In some embodiments of the foregoing, the SPECT ligand comprises amoiety selected from ¹¹¹In DOTA, ^(90Y) DOTA, ¹¹¹In, ^(90Y) and^(99m)Tc.

In other embodiments, a method of imaging in vivo target expression isprovided, the method comprising:

a) providing any of the foregoing cyclic peptides, wherein SEQ is apeptide sequence having affinity for a location on or near a targetexpressing site in a subject, and modifying the cyclic peptide toinclude a magnetic resonance ligand (MR ligand);

b) administering the cyclic peptide of step a) to the subject;

c) measuring the magnetic resonance from the MR ligand at a first time;

d) measuring the magnetic resonance from the MR ligand at a second time;and

e) comparing the magnetic resonance from the MR ligand at the first andsecond times.

In some embodiments, the MR ligand comprises Gd³⁺.

In other embodiments of the foregoing methods, the cyclic peptidecomprises a linkage to a reporter moiety, the reporter moiety selectedfrom polyethylene glycol (PEG), biotin, thiol and fluorophores. Forexample, in some embodiments the fluorophores are selected from FAM,FITC, Cy5, TRITC, TAMRA.

Disclosed herein are particular peptides that bind to CD8 (both CD8protein and CD8 on CD8+ cells). Such peptides can be used in or as acapture agent and used as a capture agent or as a binding peptide in anyof various methods for use of capture agents and binding peptides. Thesedisclosed peptides include: t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn (SEQID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh, t(CyA)(FP)kn,t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn (SEQ ID NO:4),t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6), t(CyA)ek(N-Me-a),t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQ ID NO:8),tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp (SEQ IDNO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13), tnkk(CyA) (SEQID NO:14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15), trrk(CyA) (SEQ IDNO:16), trrks (SEQ ID NO:17), tkrkr (SEQ ID NO:18), trkkh (SEQ IDNO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ ID NO:21), tshkr (SEQ IDNO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQ ID NO:24), tr(FP)kr(SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQ ID NO:27), tG(CyA)kr(SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ ID NO:29), tnpks (SEQ IDNO:31), tp(CyA)k(FP), t(CyA/e/Thz)(k/FP)k(N-Me-a/n/FP), and trrkr (SEQID NO:30).

In some forms, the peptide can be t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn(SEQ ID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh,t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn(SEQ ID NO:4), t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6),t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQID NO:8), tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp(SEQ ID NO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13),tnkk(CyA) (SEQ ID NO:14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15),trrk(CyA) (SEQ ID NO:16), trrks (SEQ ID NO:17), tkrkr (SEQ ID NO:18),trkkh (SEQ ID NO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ ID NO:21), tshkr(SEQ ID NO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQ ID NO:24),tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQ ID NO:27),tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ ID NO:29), tnpks(SEQ ID NO:31), tp(CyA)k(FP), t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), or trrkr(SEQ ID NO:30).

In some forms, the peptide can be t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn(SEQ ID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh,t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn(SEQ ID NO:4), t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6),t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQID NO:8), tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp(SEQ ID NO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13),tnkk(CyA) (SEQ ID NO:14), ts(Thz)k(CyA), or tk(FP)kk (SEQ ID NO:15).

In some forms, the peptide can be trrk(CyA) (SEQ ID NO:16), trrks (SEQID NO:17), tkrkr (SEQ ID NO:18), trkkh (SEQ ID NO:19), trnkr (SEQ IDNO:20), ttkkr (SEQ ID NO:21), tshkr (SEQ ID NO:22), t(Thz)rkk (SEQ IDNO:23), tr(Thz)kr (SEQ ID NO:24), tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQID NO:26), trGkr (SEQ ID NO:27), tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP),or te(MT)kp (SEQ ID NO:29).

In some forms, the peptide can be tnpks (SEQ ID NO:31) or tp(CyA)k(FP).In some forms, the peptide can be t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), trrkr(SEQ ID NO:30), tnpks (SEQ ID NO:31), or tp(CyA)k(FP). In some forms,the peptide can be t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), tnpks (SEQ IDNO:31), or tp(CyA)k(FP).

The disclosed compositions and methods can be further understood throughthe following numbered paragraphs.

1. A cyclic peptide having the following structure (I):

or a salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹ and L² are each individually optionally substituted linker moieties,each linker moiety optionally comprising a linkage to a solid support, alinkage to a reporter moiety, a linkage to a peptide ligand, a linkageto an azide or alkyne moiety or combinations thereof;

G is a triazole, a carbon-carbon double bond or an amide;

M is methionine;

R is H, -L³-A or —C(═O)-L³-A, where L³ is a linker moiety and A is analkyne, azide or a bond to a peptide ligand;

R¹ is H or C₁-C₆alkyl;

Y¹ and Y² are each individually 0 or 1; and

SEQ is an amino acid sequence comprising from 2 to 20 amino acidsselected from a set of amino acids, wherein the set of amino acidscomprises a combination of canonical amino acids and non-canonical aminoacids, wherein two or more of the amino acids in the set arenon-canonical amino acids and four or more of the amino acids in the setare canonical amino acids.

2. The cyclic peptide of paragraph 1, wherein G is a triazole.

3. The cyclic peptide of paragraph 1, wherein G is a carbon-carbondouble bond.

4. The cyclic peptide of any one of paragraphs 1-3, wherein L¹, L², orboth, comprise one or more substituents selected from alkyl, alkyne,azide and aminocarbonyl.

5. The cyclic peptide of any one of paragraphs 1-4, wherein L¹, L², orboth, comprise a linkage selected from a linkage to a solid support, alinkage to a reporter moiety and a linkage to a peptide ligand.

6. The cyclic peptide of any one of paragraphs 1-5, wherein L¹ and L²are alkylene.

7. The cyclic peptide of any one of paragraphs 1-6, wherein the cyclicpeptide has one of the following structures (Ia) or (Ib):

wherein:

R³ is H, a linkage to a solid support, a linkage to a reporter moiety, alinkage to a peptide ligand, a linkage to an azide or alkyne moiety orcombinations thereof; and

x and y are each independently an integer from 1 to 8.

8. The cyclic peptide of any one of paragraphs 1-7, wherein SEQcomprises from 2 to 9 amino acids.

9. The cyclic peptide of paragraph 8, wherein SEQ comprises from 5 to 7amino acids.

10. The cyclic peptide of any one of paragraphs 1-9, wherein the aminoacids are selected from Cyclopropyl Alanine (CyA), Gly, 4-FluorophenylAlanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr),4-Phenyl Phenylalanine (PhF), Asn, Ser, Thr, His, Lys, Arg, Glu,β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro.

11. The cyclic peptide of any one of paragraphs 1-9, wherein the aminoacids are selected from Cyclopropyl Alanine (CyA) and Gly;4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn, Ser, Thr; His, Lys,Arg, Glu; and N-Methyl d-alanine (N-Me-a), and Pro.

12. The cyclic peptide of any one of paragraphs 1-9, wherein the aminoacids are selected from Cyclopropyl Alanine (CyA) and Gly;4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), 2-MethoxyPyridylalanine (MeOPyr), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine(PhF), and Phe; Asn, Ser, Thr; His, Lys, Arg, Glu; and β-Phenylalanine(BPhA), N-Methyl d-alanine (N-Me-a), and Pro.

13. The cyclic peptide of any one of paragraphs 1-9, wherein SEQcomprises t(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn (SEQ ID NO:2),t(Thz)(FP)kG, tG(PhF)k(N-Me-a), t(CyA)(PhF)kh, t(CyA)(FP)kn,t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ ID NO:3), tphkn (SEQ ID NO:4),t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQ ID NO:6), t(CyA)ek(N-Me-a),t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA), tesk(CyA) (SEQ ID NO:8),tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ ID NO:10), tekkp (SEQ IDNO:11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ ID NO:13), tnkk(CyA) (SEQID NO:14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15), trrk(CyA) (SEQ IDNO:16), trrks (SEQ ID NO:17), tkrkr (SEQ ID NO:18), trkkh (SEQ IDNO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ ID NO:21), tshkr (SEQ IDNO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQ ID NO:24), tr(FP)kr(SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQ ID NO:27), tG(CyA)kr(SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ ID NO:29), tnpks (SEQ IDNO:31), tp(CyA)k(FP), t(CyA/e/Thz)(k/FP)k(N-Me-a/n/FP), or trrkr (SEQ IDNO:30).

14. The cyclic peptide of any one of paragraphs 1-13, wherein R is H or—C(═O)-L³-A, where L³ is a linker moiety and A is a bond to a peptideligand or an alkyne.

15. The cyclic peptide of paragraph 14, wherein A is an alkyne.

16. The cyclic peptide of paragraph 14, wherein A is a bond to a peptideligand.

17. The cyclic peptide of paragraph 16, wherein the peptide ligand is alinear peptide.

18. The cyclic peptide of paragraph 16, wherein the peptide ligand is acyclic peptide.

19. The cyclic peptide of paragraph 17 or 18, wherein the peptide ligandfurther comprises a second peptide ligand covalently bound thereto.

20. The cyclic peptide of any one of paragraphs 1-19, wherein y¹ and y²are each 0.

21. A composition comprising the cyclic peptide of any one of paragraphs1-20 and a pharmaceutically acceptable carrier.

22. A library comprising a plurality of cyclic peptides according to anyone of paragraphs 1-20.

23. A method for identifying a target binding compound, the methodcomprising:

(A) providing a first peptide library comprising a plurality of firstpeptide library members, the first peptide library members optionallycomprising an alkyne, azide or reporter moiety or combinations thereof;

(B) contacting the first peptide library with a target or a truncatedanalogue thereof, the target or truncated analogue thereof comprising afirst binding site and optionally an alkyne, azide or reporter moiety orcombinations thereof;

(C) identifying a first peptide library member with affinity for thefirst binding site and optionally modifying the first peptide librarymember to include an alkyne or azide moiety;

and optionally:

(D) providing a second peptide library comprising a plurality of secondpeptide library members, the second peptide library members comprisingan azide or alkyne or both;

(E) contacting the second peptide library with a composition comprisingthe target or truncated analogue thereof and the first peptide librarymember of step C;

(F) forming a triazole-linked conjugate between the first peptidelibrary member of step C and a second peptide library member, the secondpeptide library member having affinity for a second binding site on thetarget or truncated analogue thereof,

-   -   wherein the first peptide library, the second peptide library,        or both, comprise cyclic peptides comprising:        -   (i) a sequence region comprising amino and carboxy termini            and a variable peptide sequence of two to twenty amino acids            selected from a set of amino acids, wherein the set of amino            acids comprises a combination of canonical amino acids and            non-canonical amino acids, wherein two or more of the amino            acids in the set are non-canonical amino acid and four or            more of the amino acids in the set are canonical amino            acids; and        -   (ii) a linker region comprising a α-amino carbonyl, α-amido            carbonyl, a methionine amino acid, or combinations thereof,            and optionally comprising an alkyne, an azide, a linkage to            a solid support or a linkage to a reporter moiety or a            combination thereof, the linker region covalently linking            the amino and carboxy termini of the sequence region.

24. The method of paragraph 23, wherein the linker region comprises aα-amino carbonyl group bound to the amino terminus of the peptidesequence.

25. The method of paragraph 24, further comprising determining thepeptide sequence of one or more of the cyclic peptides by Edmandegradation.

26. The method of paragraph 25, wherein the linker region comprises amethionine amino acid bound to the amino terminus of the variablepeptide sequence.

27. The method of paragraph 26 further comprising treating one or moreof the cyclic peptides with CNBr and determining the sequence thereof bymass spectrometry.

28. The method of paragraph 23, wherein the linker region comprises analkyne or azide, the target or a truncated analogue thereof comprises analkyne or azide and identifying the first peptide library member withaffinity for the first binding site comprises identifying first peptidelibrary members which form a triazole linkage with the target or atruncated analogue thereof.

29. The method of any one of paragraphs 23-28, wherein the first peptidelibrary is contacted with a truncated analogue of the target.

30. The method of any one of paragraphs 23-29, further comprisingmodifying the triazole linked conjugate to contain a triazole or alkyneand contacting the modified conjugate with the target or truncatedanalogue thereof and a third peptide library, the third peptide librarycomprising a plurality of third peptide library members, each thirdpeptide library member comprising an azide or alkyne.

31. The method of paragraph 30, further comprising forming a triazolelinkage between the modified conjugate and a member of the third peptidelibrary, the third peptide library member having affinity for a thirdbinding site on the target or truncated analogue thereof.

32. The method of any one of paragraphs 23-31, wherein the first bindingsite is an epitope.

33. The method of any one of paragraphs 23-32, wherein the secondbinding site is an epitope.

34. The method of any one of paragraphs 23-33, wherein the third bindingsite is an epitope.

35. The method of any one of paragraphs 23-34, wherein the linker regioncomprises a carbon-carbon double bond or a triazole.

36. The method of any one of paragraphs 23-35, wherein the cyclicpeptides have the following structure (I′):

or a salt, tautomer or stereoisomer thereof, wherein:

L¹ and L² are each individually optionally substituted linker moieties,each linker moiety optionally comprising a linkage to a solid support, alinkage to a reporter moiety, a linkage to an azide or alkyne moiety orcombinations thereof;

G is a triazole a carbon-carbon double bond or an amide;

M is methionine;

R is H, -L³-A or —C(═O)-L³-A, where L³ is a linker moiety and A is analkyne or azide;

R¹ is H or C₁-C₆alkyl;

Y¹ and Y² are each individually 0 or 1; and

SEQ is the variable peptide sequence.

37. The method of paragraph 36, wherein SEQ comprises from 2 to 9 aminoacids.

38. The method of paragraph 37, wherein SEQ comprises from 5 to 7 aminoacids.

39. The method of any one of paragraphs 26-38, wherein the amino acidsare selected from Cyclopropyl Alanine (CyA), Gly, 4-Fluorophenyl Alanine(FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr),4-Phenyl Phenylalanine (PhF), Asn, Ser, Thr, His, Lys, Arg, Glu,β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro.

40. The method of any one of paragraphs 26-38, wherein the amino acidsare selected from Cyclopropyl Alanine (CyA) and Gly; 4-FluorophenylAlanine (FP), Methyl Tryptophan (MT), Thiazolyl Alanine (Thz), 4-PhenylPhenylalanine (PhF), and Phe; Asn, Ser, Thr; His, Lys, Arg, Glu; andN-Methyl d-alanine (N-Me-a), and Pro.

41. The method of any one of paragraphs 26-38, wherein the amino acidsare selected from Cyclopropyl Alanine (CyA) and Gly; 4-FluorophenylAlanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr),Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn,Ser, Thr; His, Lys, Arg, Glu; and β-Phenylalanine (BPhA), N-Methyld-alanine (N-Me-a), and Pro.

42. The method of any one of paragraphs 26-38, wherein SEQ comprisest(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn (SEQ ID NO:2), t(Thz)(FP)kG,tG(PhF)k(N-Me-a), t(CyA)(PhF)kh, t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn,t(N-Me-a)pke (SEQ ID NO:3), tphkn (SEQ ID NO:4), t(CyA)rks (SEQ IDNO:5), tpkk(N-Me-a) (SEQ ID NO:6), t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ IDNO:7), t(CyA)tk(CyA), tesk(CyA) (SEQ ID NO:8), tetk(N-Me-a) (SEQ IDNO:9), tenk(FP) (SEQ ID NO:10), tekkp (SEQ ID NO:11), tskk(N-Me-a) (SEQID NO:12), ttrk (SEQ ID NO:13), tnkk(CyA) (SEQ ID NO:14), ts(Thz)k(CyA),tk(FP)kk (SEQ ID NO:15), trrk(CyA) (SEQ ID NO:16), trrks (SEQ ID NO:17),tkrkr (SEQ ID NO:18), trkkh (SEQ ID NO:19), trnkr (SEQ ID NO:20), ttkkr(SEQ ID NO:21), tshkr (SEQ ID NO:22), t(Thz)rkk (SEQ ID NO:23),tr(Thz)kr (SEQ ID NO:24), tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQ IDNO:26), trGkr (SEQ ID NO:27), tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP),te(MT)kp (SEQ ID NO:29), tnpks (SEQ ID NO:31), tp(CyA)k(FP),t(CyA/e/Thz)(k/FP)k(N-Me-a/n/FP), or trrkr (SEQ ID NO:30).

43. The method of any one of paragraphs 23-42, wherein the cyclicpeptides have the following structure (I′a):

wherein:

L^(1a) is a linker moiety optionally substituted with one or moresubstituents selected from a linkage to an alkyne or azide moiety, alinkage to a solid support and a linkage to a reporter moiety.

44. The method of paragraph 43, wherein L¹⁸ and L² are eachindependently optionally substituted alkylene.

45. The method of any one of paragraphs 23-44, wherein the cyclicpeptides have the following structure (I′b):

wherein:

R³ is H, a linkage to a solid support, a linkage to an alkyne or azidemoiety or a linkage to a reporter moiety;

R⁴ is H or C₁-C₆alkyl; and

x and y are each independently integers from 1 to 8.

46. The method of any one of paragraphs 36-45, wherein G is a triazole.

47. The method of any one of paragraphs 36-45, wherein G is acarbon-carbon double bond.

48. The method of paragraph 36, wherein the cyclic peptides have one ofthe following structures:

49. The method of any one of paragraphs 23-48, wherein the first peptidelibrary comprises the cyclic peptides, and the second peptide librarycomprises linear peptide members.

50. The method of any one of paragraphs 23-48, wherein the secondpeptide library comprises the cyclic peptides, and the first peptidelibrary comprises linear peptide members.

51. The method of any one of paragraphs 23-48, wherein the first andsecond peptide library comprises the cyclic peptides.

52. The method of any one of paragraphs 23-48, wherein the target is aprotein.

53. The method of paragraph 52, wherein the protein is an enzyme or cellsurface protein.

54. A method of detecting a target protein in a sample, the methodcomprising replacing an antibody or its equivalent in a cell-based or animmunoassay with the cyclic peptide of any one of paragraphs 1-20.

55. The method of paragraph 54, wherein the immunoassay is a Westernblot, a pull-down assay, a dot blot or an ELISA.

56. A method for inhibiting activity of a protein in a subject, themethod comprising administering an effective amount of the cyclicpeptide of any one of paragraphs 1-20 to a subject in need thereof.

57. A method of purifying a target, the method comprising immobilizingthe cyclic peptide of any one of paragraphs 1-20 in a column basedformat, contacting the column with a matrix containing the target,washing the column, and eluting the target.

58. A method of imaging in vivo target expression, the methodcomprising:

-   -   (a) providing a cyclic peptide of any one of paragraphs 1-20,        wherein SEQ is a peptide sequence having affinity for a location        on or near a target expressing site in a subject, and modifying        the cyclic peptide to include a small-molecule        positron-emission-tomography ligand (PET ligand);    -   (b) administering the cyclic peptide of step (a) to the subject;    -   (c) measuring the positron emission from the PET ligand at a        first time;    -   (d) measuring the positron emission from the PET ligand at a        second time; and    -   (e) comparing the positron emission from the PET ligand at the        first and second times.

59. The method of paragraph 58, wherein the PET ligand comprises amoiety selected from ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ⁶⁸Ga NOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga,⁸⁹Zr, ₁₂₄I, ⁸⁶Y, ^(94m)Tc, ^(110m)In, ¹¹C and ⁷⁶Br.

60. A method of imaging in vivo target expression, the methodcomprising:

(a) providing a cyclic peptide of any one of paragraphs 1-20, whereinSEQ is a peptide sequence having affinity for a location on or near atarget expressing site in a subject, and modifying the cyclic peptide toinclude a small-molecule single-photon-emission-computed-tomographyligand (SPECT ligand);

(b) administering the cyclic peptide of step (a) to the subject;

(c) measuring the photon emission from the SPECT ligand at a first time;

(d) measuring the photon emission from the SPECT ligand at a secondtime; and

(e) comparing the photon emission from the SPECT ligand at the first andsecond times.

61. The method of paragraph 60, wherein the SPECT ligand comprises amoiety selected from ¹¹In DOTA, ^(90Y) DOTA, ¹¹¹In, ⁹⁰Y and ^(99m)Tc.

62. A method of imaging in vivo target expression, the methodcomprising:

(a) providing a cyclic peptide of any one of paragraphs 1-20, whereinSEQ is a peptide sequence having affinity for a location on or near atarget expressing site in a subject, and modifying the cyclic peptide toinclude a magnetic resonance ligand (MR ligand);

(b) administering the cyclic peptide of step (a) to the subject;

(c) measuring the magnetic resonance from the MR ligand at a first time;

(d) measuring the magnetic resonance from the MR ligand at a secondtime; and

(e) comparing the magnetic resonance from the MR ligand at the first andsecond times.

63. The method of paragraph 62, wherein the MR ligand comprises Gd³⁺.

64. The cyclic peptide of any one of paragraphs 1-20 or the method ofany one of paragraphs 26-63, wherein the cyclic peptide comprises alinkage to a reporter moiety, the reporter moiety selected frompolyethylene glycol (PEG), biotin, thiol and fluorophores.

65. The cyclic peptide or method of paragraph 64, wherein thefluorophores are selected from carboxyfluorescein (FAM), fluoresceinisothiocyanate (FITC), Cyanine-5 (Cy5), tetramethylrhodamine (TRITC) andCarboxytetramethylrhodamine (TAMRA).

EXAMPLES Example 1: Synthesis of Ring Closing Metathesis (RCM) CatalyzedOBOC Peptide Libraries

A linear library of the form Fmoc-R-I₈-G-X₁X₂X₃X₄X₅-S-I₅-TG,(R-I₈=R-2-amino-2methyldec-9-enoic acid), TG=Tentagel-S—NH₂ resin,S-I₈=S-2-amino-2methyldec-9-enoic acid, and Xi=D-Ala, D-Arg, D-Asn,D-Asp, D-Glu, D-Gln, Gly, D-His, D-Ile, D-Leu, D-Lys, D-Phe, D-Pro,D-D-Thr, D-Trp, D-Tyr, D-Val) was synthesized using standard solid phasetechniques. The amino acid after S-I₅ was double coupled using standardSPPS protocols, to maintain the efficiency of the coupling step. Theamino acid pair R-I₈ and S-I₅ were chosen for the RCM reaction, sinceR-I₈ and S-I₅, separated by 6 amino acids in a helical peptide, providesvery effective crosslinking (98% yield). While a majority of therandomized OBOC peptides will not be of a helical form, the long alkylcross linker (total 13 atoms) should enable the peptides to cyclize onbead via RCM. The on bead peptides were cyclized using Grubb's catalyst,bis(tricyclohexylphosphine)benzylidine ruthenium(IV) dichlorideaccording to literature methods. Briefly, in an argon atmosphere, thepeptide library R-I₈-G-X₁X₂X₃X₄X₅-S-I₅-TG was swelled for 30 minutes inanhydrous dichloroethane solution. 1 ml of 10 mM solution of the Grubb'scatalyst in dichloroethane was added to the swelled resin and thereaction was allowed to proceed for 6 hours. The solution was drainedand the reaction with the catalyst was repeated again for 6 hours. Thelibrary was washed with anhydrous dichloroethane and the excess catalystwas removed using oxine in DMF. The Fmoc group was removed by treatmentwith 20% piperidine/NMP. The library was washed and dried and the sidechains were deprotected by treatment with 95:2.5:2.5 TFA/TES/H₂O. Thefinal cyclized library was stored in TBS buffer.

Example 2: Synthesis of Triazole Cyclized OBOC Libraries

The linear peptide libraries A, B, C were synthesized on Tentagel-S—NH₂resin using standard SPPS library synthesis methods. Libraries B and Cwere synthesized at a 10-fold excess to ensure adequate representationof each library element. After the synthesis of each library, all thebeads in the linear library were subjected to an on bead CuAAC reaction,for 6 hours at room temperature with 1.5 equivalents of CuI, 2.5equivalents of ascorbic acid in 20% piperidine in DMF. After washes with10% sodium diethyldithiocarbamate in DMF to remove the adsorbed Cu, thelibrary was washed with DMF, methanol and DCM and dried. Random beadswere picked from the library to be sequenced. The rest of the librarywas stored in NMP until used.

Example 3: Synthesis of Triazole Cyclized OBOC Libraries

A panel of D and L non-canonical amino acids were obtained to enablestandard solid phase peptide synthesis of OBOC libraries withdiversified side chains (Table 2). These new amino acids incorporateadditional steric compression (methylated amino acids), electronicallymodulated phenyl substituents, and various heterocycles. Homologatedamino acids including beta amino acids were included to add additionalflexibility to the peptide macrocycle. Finally, natural amino acids withmetabolic liabilities (i.e. tyrosine and tryptophan) were replaced withrobust derivatives. Test sequences were prepared to ensure that theunnatural amino acid could be incorporated using standard peptidecoupling conditions during the split/mix library synthesis. These testsequences were also sequenced by MALDI-TOF/TOF mass spectrometry toensure the amino acids were sequenceable.

TABLE 2 Panel of Non-Canonical Amino Acids

Several of these non-canonical amino acids were included in a focusedlibrary (Table 4; Set 2) to improve a previous hit, tfpkk (SEQ ID NO:1). The threonine in the first position and lysine in the fourthposition were found to be critical for target binding (CD8). The threeother positions sampled the 16 amino acids listed in Table 4, randomlyincluding one of the 16 amino acids at each of the second, third, andfifth positions. Tyrosine was removed from the library and replaced withadditional aromatic non-canonical amino acids including 4-fluorophenylalanine and 4-phenyl phenylalanine. Tryptophan's metabolic liability wasaddressed by methylation of the indole N—H. The thiazolyl derivative isa histidine derivative, replacing the hydrogen bond donation ofhistidine with a polarizable thio-heterocycle. Cyclopropyl alanine, acyclic derivative of valine was included to sample this compressedderivative. An N-methylated amino acid, N-methylalanine was included toprovide hits with enhanced flexibility. Together, the inclusion ofnon-canonical amino acids greatly increased the chemical diversity andbioactivity of the resulting peptides. This library has 16³=4096 uniquespecies (1 g=700 copies).

TABLE 4 Focused Library including Non-Canonical Amino Acids HydrophobicSide Chain-Aliphatic

Hydrophobic Side Chain-Aromatic

Polar Side Chain-Neutral

Polar Side Chain-Charged

Conformational Perturbation

This focused library entered into a screen to identify enhanced CD8binders. Non-specific PCCs were identified and removed from the libraryby performing an anti-screen against the glycosylated cell-surfaceprotein PSMA. The enriched library was then screened against CD8a,yielding 23 hits (Table 5). These hits display good homology, suggestingtractable structure-activity-relationship among the hits. Eachnon-canonical amino acid was represented at least once among the hits,with the exception of 1-methyltryptophan. The consensus sequence fromthe hits in Table 5 is t(CyA/e/Thz)(k/FP)k(N-Me-a/n/FP).

TABLE 5 Hit Sequences Following CD8 Protein Screen x1 x2 x3 x4 x5 SEQ IDNO t Thz p k FP PhF G FP Thz h n 2 Thz FP G G PhF N-Me-a CyA PhF h CyAFP n N-Me-a N-Me-a n N-Me-a p e 3 p h n 4 CyA r s 5 p k N-Me-a 6 CyA eN-Me-a CyA e h 7 CyA t CyA e s CyA 8 e t N-Me-a 9 e n FP 10 e k p 11 s kN-Me-1 12 t r — 13 n k CyA 14 s Thz CyA k FP k 15

In addition to screening the focused library against recombinant humanCD8 protein, the library was screened against cells. Non-specific PCCswere identified and removed by an anti-screen against CD8− Jurkat cells.The pre-cleared library entered into a screen against CD8+ SupT1 cellsin 2% FBS for 1 h at 4° C. The resulting hits (Table 6) showed a highlevel of sequence homology, with electrostatic side chains dominatingthe hit profile. Non-canonical amino acids including cyclopropylalanine, thiazolyl alanine, 4-fluorophenylalanine, and1-methyltryptophan were identified among the hits. The consensussequence from the hits in Table 6 is trrkr (SEQ ID NO:30).

Because of the large number of electrostatic hits in the screen with 2%FBS, additional screens were performed varying the blocking conditionsto decrease the quantity of electrostatic hits. In particular, screensusing 10% FBS gave no hits with any electrostatic amino acids. Theseconditions ultimately identified tnpks (SEQ ID NO:31) and tp(CyA)k(Fp).

TABLE 6 Hit Sequences Following CD8+ Cell Screen x1 x2 x3 x4 x5 SEQ IDNO t r r k CyA 16 t r r k s 17 t k r k r 18 t r k k h 19 t r n k r 20 tt k k r 21 t s h k r 22 t Thz r k k 23 t r Thz k r 24 t r FP k r 25 t kFP k r 26 t r G k r 27 t G CyA k r 28 t p CyA k FP t e MT k P 29

The hits identified in the non-canonical screen were further evaluatedin CD8+ SupT1 and CD8− Jurkat cells by flow cytometry. Each hitdisplayed preferential binding to the CD8+ cell line. Additionally,tp(CyA)k(FP) demonstrated improved binding to CD8 over the parentcompound tfpkk (SEQ ID NO:1) (FIG. 1).

Any of these identified peptides can be used in or as a capture agentand used as a capture agent or as a binding peptide in any of variousmethods for use of capture agents and binding peptides.

It is understood that the disclosed method and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a peptide is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the peptide are discussed, each and every combination andpermutation of peptide and the modifications that are possible arespecifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as aclass of molecules D, E, and F and an example of a combination molecule,A-D is disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. Further, each of the materials, compositions,components, etc. contemplated and disclosed as above can also bespecifically and independently included or excluded from any group,subgroup, list, set, etc. of such materials. These concepts apply to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed compositions. Thus, if thereare a variety of additional steps that can be performed it is understoodthat each of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods, andthat each such combination is specifically contemplated and should beconsidered disclosed.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apeptide” includes a plurality of such peptides, reference to “thepeptide” is a reference to one or more peptides and equivalents thereofknown to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Unless the context clearly indicates otherwise, use of the word “can”indicates an option or capability of the object or condition referredto. Generally, use of “can” in this way is meant to positively state theoption or capability while also leaving open that the option orcapability could be absent in other forms or embodiments of the objector condition referred to. Unless the context clearly indicatesotherwise, use of the word “may” indicates an option or capability ofthe object or condition referred to. Generally, use of “may” in this wayis meant to positively state the option or capability while also leavingopen that the option or capability could be absent in other forms orembodiments of the object or condition referred to. Unless the contextclearly indicates otherwise, use of “may” herein does not refer to anunknown or doubtful feature of an object or condition.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. It shouldbe understood that all of the individual values and sub-ranges of valuescontained within an explicitly disclosed range are also specificallycontemplated and should be considered disclosed unless the contextspecifically indicates otherwise. Finally, it should be understood thatall ranges refer both to the recited range as a range and as acollection of individual numbers from and including the first endpointto and including the second endpoint. In the latter case, it should beunderstood that any of the individual numbers can be selected as oneform of the quantity, value, or feature to which the range refers. Inthis way, a range describes a set of numbers or values from andincluding the first endpoint to and including the second endpoint fromwhich a single member of the set (i.e. a single number) can be selectedas the quantity, value, or feature to which the range refers. Theforegoing applies regardless of whether in particular cases some or allof these embodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart.

The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Although the description of materials, compositions, components, steps,techniques, etc. can include numerous options and alternatives, thisshould not be construed as, and is not an admission that, such optionsand alternatives are equivalent to each other or, in particular, areobvious alternatives. Thus, for example, a list of different amino acidsdoes not indicate that the listed amino acids are obvious one to theother, nor is it an admission of equivalence or obviousness.

Every peptide disclosed herein is intended to be and should beconsidered to be specifically disclosed herein. Further, every subgroupthat can be identified within this disclosure is intended to be andshould be considered to be specifically disclosed herein. As a result,it is specifically contemplated that any peptide, or subgroup ofpeptides can be either specifically included for or excluded from use orincluded in or excluded from a list of peptides.

Those skilled in the art will recognize many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

We claim:
 1. A cyclic peptide having the following structure (I):

or a salt, tautomer, prodrug or stereoisomer thereof, wherein: L¹ and L²are each individually optionally substituted linker moieties, eachlinker moiety optionally comprising a linkage to a solid support, alinkage to a reporter moiety, a linkage to a peptide ligand, a linkageto an azide or alkyne moiety or combinations thereof; G is a triazole, acarbon-carbon double bond or an amide; M is methionine; R is H, -L³-A or—C(═O)-L³-A, where L³ is a linker moiety and A is an alkyne, azide or abond to a peptide ligand; R¹ is H or C₁-C₆alkyl; Y¹ and Y² are eachindividually 0 or I; and SEQ is an amino acid sequence comprising from 2to 20 amino acids selected from a set of amino acids, wherein the set ofamino acids comprises a combination of canonical amino acids andnon-canonical amino acids, wherein two or more of the amino acids in theset are non-canonical amino acids and four or more of the amino acids inthe set are canonical amino acids.
 2. The cyclic peptide of claim 1,wherein G is a triazole.
 3. The cyclic peptide of claim 1, wherein G isa carbon-carbon double bond.
 4. The cyclic peptide of claim 1, whereinL¹, L², or both, comprise one or more substituents selected from alkyl,alkyne, azide and aminocarbonyl.
 5. The cyclic peptide of claim 1,wherein L¹, L², or both, comprise a linkage selected from a linkage to asolid support, a linkage to a reporter moiety and a linkage to a peptideligand.
 6. The cyclic peptide of claim 1, wherein L¹ and L² arealkylene.
 7. The cyclic peptide of claim 1, wherein the cyclic peptidehas one of the following structures (Ia) or (Ib):

wherein: R³ is H, a linkage to a solid support, a linkage to a reportermoiety, a linkage to a peptide ligand, a linkage to an azide or alkynemoiety or combinations thereof; and x and y are each independently aninteger from 1 to
 8. 8. The cyclic peptide of claim 1, wherein SEQcomprises from 2 to 9 amino acids.
 9. The cyclic peptide of claim 8,wherein SEQ comprises from 5 to 7 amino acids.
 10. The cyclic peptide ofclaim 1, wherein the amino acids are selected from Cyclopropyl Alanine(CyA), Gly, 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT),2-Methoxy Pyridylalanine (MeOPyr), 4-Phenyl Phenylalanine (PhF), Asn,Ser, Thr, His, Lys, Arg, Glu, β-Phenylalanine (BPhA), N-Methyl d-alanine(N-Me-a), and Pro.
 11. The cyclic peptide of claim 1, wherein the aminoacids are selected from Cyclopropyl Alanine (CyA) and Gly;4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn, Ser, Thr; His, Lys,Arg, Glu; and N-Methyl d-alanine (N-Me-a), and Pro.
 12. The cyclicpeptide of claim 1, wherein the amino acids are selected fromCyclopropyl Alanine (CyA) and Gly; 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn, Ser, Thr, His, Lys,Arg, Glu; and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), andPro.
 13. The cyclic peptide of claim 1, wherein SEQ comprisest(Thz)pk(FP), t(PhF)Gk(FP), t(Thz)hkn (SEQ ID NO:2), t(Thz)(FP)kG,tG(PhF)k(N-Me-a), t(CyA)(PhF)kh, t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn,t(N-Me-a)pke (SEQ ID NO:3), tphkn (SEQ ID NO:4), t(CyA)rks (SEQ IDNO:5), tpkk(N-Me-a) (SEQ ID NO:6), t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ IDNO:7), t(CyA)tk(CyA), tesk(CyA) (SEQ ID NO:8), tetk(N-Me-a) (SEQ IDNO:9), tenk(FP) (SEQ ID NO: 10), tekkp (SEQ ID NO: 11), tskk(N-Me-a)(SEQ ID NO: 12), ttrk (SEQ ID NO:13), tnkk(CyA) (SEQ ID NO:14),ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15), trrk(CyA) (SEQ ID NO:16), trrks(SEQ ID NO:17), tkrkr (SEQ ID NO:18), trkkh (SEQ ID NO:19), trnkr (SEQID NO:20), ttkkr (SEQ ID NO:21), tshkr (SEQ ID NO:22), t(Thz)rkk (SEQ IDNO:23), tr(Thz)kr (SEQ ID NO:24), tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQID NO:26), trGkr (SEQ ID NO:27), tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP),te(MT)kp (SEQ ID NO:29), tnpks (SEQ ID NO:31), tp(CyA)k(FP),t(CyA/e/Thz)(k/FP)k(N-Me-a/n/FP), or trrkr (SEQ ID NO:30).
 14. Thecyclic peptide of claim 1, wherein R is H or —C(═O)-L³-A, where L³ is alinker moiety and A is a bond to a peptide ligand or an alkyne.
 15. Thecyclic peptide of claim 14, wherein A is an alkyne.
 16. The cyclicpeptide of claim 14, wherein A is a bond to a peptide ligand.
 17. Thecyclic peptide of claim 16, wherein the peptide ligand is a linearpeptide.
 18. The cyclic peptide of claim 16, wherein the peptide ligandis a cyclic peptide.
 19. The cyclic peptide of claim 17, wherein thepeptide ligand further comprises a second peptide ligand covalentlybound thereto.
 20. The cyclic peptide of claim 1, wherein y¹ and y² areeach
 0. 21. A composition comprising the cyclic peptide of claim 1 and apharmaceutically acceptable carrier.
 22. A library comprising aplurality of cyclic peptides according to claim
 1. 23. A method foridentifying a target binding compound, the method comprising: (A)providing a first peptide library comprising a plurality of firstpeptide library members, the first peptide library members optionallycomprising an alkyne, azide or reporter moiety or combinations thereof;(B) contacting the first peptide library with a target or a truncatedanalogue thereof, the target or truncated analogue thereof comprising afirst binding site and optionally an alkyne, azide or reporter moiety orcombinations thereof; (C) identifying a first peptide library memberwith affinity for the first binding site and optionally modifying thefirst peptide library member to include an alkyne or azide moiety; andoptionally: (D) providing a second peptide library comprising aplurality of second peptide library members, the second peptide librarymembers comprising an azide or alkyne or both; (E) contacting the secondpeptide library with a composition comprising the target or truncatedanalogue thereof and the first peptide library member of step C; (F)forming a triazole-linked conjugate between the first peptide librarymember of step C and a second peptide library member, the second peptidelibrary member having affinity for a second binding site on the targetor truncated analogue thereof, wherein the first peptide library, thesecond peptide library, or both, comprise cyclic peptides comprising:(i) a sequence region comprising amino and carboxy termini and avariable peptide sequence of two to twenty amino acids selected from aset of amino acids, wherein the set of amino acids comprises acombination of canonical amino acids and non-canonical amino acids,wherein two or more of the amino acids in the set are non-canonicalamino acid and four or more of the amino acids in the set are canonicalamino acids; and (ii) a linker region comprising a α-amino carbonyl,α-amido carbonyl, a methionine amino acid, or combinations thereof, andoptionally comprising an alkyne, an azide, a linkage to a solid supportor a linkage to a reporter moiety or a combination thereof, the linkerregion covalently linking the amino and carboxy termini of the sequenceregion.
 24. The method of claim 23, wherein the linker region comprisesa α-amino carbonyl group bound to the amino terminus of the peptidesequence.
 25. The method of claim 24, further comprising determining thepeptide sequence of one or more of the cyclic peptides by Edmandegradation.
 26. The method of claim 25, wherein the linker regioncomprises a methionine amino acid bound to the amino terminus of thevariable peptide sequence.
 27. The method of claim 26 further comprisingtreating one or more of the cyclic peptides with CNBr and determiningthe sequence thereof by mass spectrometry.
 28. The method of claim 23,wherein the linker region comprises an alkyne or azide, the target or atruncated analogue thereof comprises an alkyne or azide and identifyingthe first peptide library member with affinity for the first bindingsite comprises identifying first peptide library members which form atriazole linkage with the target or a truncated analogue thereof. 29.The method of claim 23, wherein the first peptide library is contactedwith a truncated analogue of the target.
 30. The method of claim 23,further comprising modifying the triazole linked conjugate to contain atriazole or alkyne and contacting the modified conjugate with the targetor truncated analogue thereof and a third peptide library, the thirdpeptide library comprising a plurality of third peptide library members,each third peptide library member comprising an azide or alkyne.
 31. Themethod of claim 30, further comprising forming a triazole linkagebetween the modified conjugate and a member of the third peptidelibrary, the third peptide library member having affinity for a thirdbinding site on the target or truncated analogue thereof.
 32. The methodof claim 23, wherein the first binding site is an epitope.
 33. Themethod of claim 23, wherein the second binding site is an epitope. 34.The method of claim 23, wherein the third binding site is an epitope.35. The method of claim 23, wherein the linker region comprises acarbon-carbon double bond or a triazole.
 36. The method of claim 23,wherein the cyclic peptides have the following structure (I′):

or a salt, tautomer or stereoisomer thereof, wherein: L¹ and L² are eachindividually optionally substituted linker moieties, each linker moietyoptionally comprising a linkage to a solid support, a linkage to areporter moiety, a linkage to an azide or alkyne moiety or combinationsthereof; G is a triazole a carbon-carbon double bond or an amide; M ismethionine; R is H, -L³-A or —C(═O)-L³-A, where L³ is a linker moietyand A is an alkyne or azide; R¹ is H or C₁-C₆alkyl; Y¹ and Y² are eachindividually 0 or 1; and SEQ is the variable peptide sequence.
 37. Themethod of claim 36, wherein SEQ comprises from 2 to 9 amino acids. 38.The method of claim 37, wherein SEQ comprises from 5 to 7 amino acids.39. The method of claim 26, wherein the amino acids are selected fromCyclopropyl Alanine (CyA), Gly, 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), 4-PhenylPhenylalanine (PhF), Asn, Ser, Thr, His, Lys, Arg, Glu, β-Phenylalanine(BPhA), N-Methyl d-alanine (N-Me-a), and Pro.
 40. The method of claim26, wherein the amino acids are selected from Cyclopropyl Alanine (CyA)and Gly; 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), ThiazolylAlanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn, Ser, Thr;His, Lys, Arg, Glu; and N-Methyl d-alanine (N-Me-a), and Pro.
 41. Themethod of claim 26, wherein the amino acids are selected fromCyclopropyl Alanine (CyA) and Gly; 4-Fluorophenyl Alanine (FP), MethylTryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine(Thz), 4-Phenyl Phenylalanine (PhF), and Phe; Asn, Ser, Thr; His, Lys,Arg, Glu; and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), andPro.
 42. The method of claim 26, wherein SEQ comprises t(Thz)pk(FP),t(PhF)Gk(FP), t(Thz)hkn (SEQ ID NO:2), t(Thz)(FP)kG, tG(PhF)k(N-Me-a),t(CyA)(PhF)kh, t(CyA)(FP)kn, t(N-Me-a)(N-Me-a)kn, t(N-Me-a)pke (SEQ IDNO:3), tphkn (SEQ ID NO:4), t(CyA)rks (SEQ ID NO:5), tpkk(N-Me-a) (SEQID NO:6), t(CyA)ek(N-Me-a), t(CyA)ekh (SEQ ID NO:7), t(CyA)tk(CyA),tesk(CyA) (SEQ ID NO:8), tetk(N-Me-a) (SEQ ID NO:9), tenk(FP) (SEQ IDNO:10), tekkp (SEQ ID NO: 11), tskk(N-Me-a) (SEQ ID NO:12), ttrk (SEQ IDNO:13), tnkk(CyA) (SEQ ID NO: 14), ts(Thz)k(CyA), tk(FP)kk (SEQ ID NO:15), trrk(CyA) (SEQ ID NO:16), trrks (SEQ ID NO:17), tkrkr (SEQ IDNO:18), trkkh (SEQ ID NO:19), trnkr (SEQ ID NO:20), ttkkr (SEQ IDNO:21), tshkr (SEQ ID NO:22), t(Thz)rkk (SEQ ID NO:23), tr(Thz)kr (SEQID NO:24), tr(FP)kr (SEQ ID NO:25), tk(FP)kr (SEQ ID NO:26), trGkr (SEQID NO:27), tG(CyA)kr (SEQ ID NO:28), tp(CyA)k(FP), te(MT)kp (SEQ IDNO:29), tnpks (SEQ ID NO:31), tp(CyA)k(FP),t(CyA/e/ThzXk/FP)k(N-Me-a/n/FP), or trrkr (SEQ ID NO:30).
 43. The methodof claim 23, wherein the cyclic peptides have the following structure(I′a):

wherein: L^(1a) is a linker moiety optionally substituted with one ormore substituents selected from a linkage to an alkyne or azide moiety,a linkage to a solid support and a linkage to a reporter moiety.
 44. Themethod of claim 43, wherein L^(1a) and L² are each independentlyoptionally substituted alkylene.
 45. The method of claim 23, wherein thecyclic peptides have the following structure (I′b):

wherein: R³ is H, a linkage to a solid support, a linkage to an alkyneor azide moiety or a linkage to a reporter moiety; R⁴ is H orC₁-C₆alkyl; and x and y are each independently integers from 1 to
 8. 46.The method of claim 36, wherein G is a triazole.
 47. The method of claim36, wherein G is a carbon-carbon double bond.
 48. The method of claim36, wherein the cyclic peptides have one of the following structures:


49. The method of claim 23, wherein the first peptide library comprisesthe cyclic peptides, and the second peptide library comprises linearpeptide members.
 50. The method of claim 23, wherein the second peptidelibrary comprises the cyclic peptides, and the first peptide librarycomprises linear peptide members.
 51. The method of claim 23, whereinthe first and second peptide library comprises the cyclic peptides. 52.The method of claim 23, wherein the target is a protein.
 53. The methodof claim 52, wherein the protein is an enzyme or cell surface protein.54. A method of detecting a target protein in a sample, the methodcomprising replacing an antibody or its equivalent in a cell-based or animmunoassay with the cyclic peptide of claim
 1. 55. The method of claim54, wherein the immunoassay is a Western blot, a pull-down assay, a dotblot or an ELISA.
 56. A method for inhibiting activity of a protein in asubject, the method comprising administering an effective amount of thecyclic peptide of claim 1 to a subject in need thereof.
 57. A method ofpurifying a target, the method comprising immobilizing the cyclicpeptide of claim 1 in a column based format, contacting the column witha matrix containing the target, washing the column, and eluting thetarget.
 58. A method of imaging in vivo target expression, the methodcomprising: (a) providing a cyclic peptide of claim 1, wherein SEQ is apeptide sequence having affinity for a location on or near a targetexpressing site in a subject, and modifying the cyclic peptide toinclude a small-molecule positron-emission-tomography ligand (PETligand); (b) administering the cyclic peptide of step (a) to thesubject; (c) measuring the positron emission from the PET ligand at afirst time; (d) measuring the positron emission from the PET ligand at asecond time; and (e) comparing the positron emission from the PET ligandat the first and second times.
 59. The method of claim 58, wherein thePET ligand comprises a moiety selected from ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ⁶⁸GaNOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴I, ⁸⁶Y, ^(94m)Tc, ^(110m)In, ¹¹C and⁷⁶Br.
 60. A method of imaging in vivo target expression, the methodcomprising: (a) providing a cyclic peptide of claim 1, wherein SEQ is apeptide sequence having affinity for a location on or near a targetexpressing site in a subject, and modifying the cyclic peptide toinclude a small-molecule single-photon-emission-computed-tomographyligand (SPECT ligand); (b) administering the cyclic peptide of step (a)to the subject; (c) measuring the photon emission from the SPECT ligandat a first time; (d) measuring the photon emission from the SPECT ligandat a second time; and (e) comparing the photon emission from the SPECTligand at the first and second times.
 61. The method of claim 60,wherein the SPECT ligand comprises a moiety selected from ¹¹¹In DOTA,90Y DOTA, ¹¹¹In, ⁹⁰Y and ^(99m)Tc.
 62. A method of imaging in vivotarget expression, the method comprising: (a) providing a cyclic peptideof claim 1, wherein SEQ is a peptide sequence having affinity for alocation on or near a target expressing site in a subject, and modifyingthe cyclic peptide to include a magnetic resonance ligand (MR ligand);(b) administering the cyclic peptide of step (a) to the subject; (c)measuring the magnetic resonance from the MR ligand at a first time; (d)measuring the magnetic resonance from the MR ligand at a second time;and (e) comparing the magnetic resonance from the MR ligand at the firstand second times.
 63. The method of claim 62, wherein the MR ligandcomprises Gd³⁺.
 64. The cyclic peptide of claim 1, wherein the cyclicpeptide comprises a linkage to a reporter moiety, the reporter moietyselected from polyethylene glycol (PEG), biotin, thiol and fluorophores.65. The cyclic peptide of claim 64, wherein the fluorophores areselected from carboxyfluorescein (FAM), fluorescein isothiocyanate(FITC), Cyanine-5 (Cy5), tetramethylrhodamine (TRITC) andCarboxytetramethylrhodamine (TAMRA).